Hepatozoon parasite was initially reported from a cat in India in 1908 and named Leucocytozoon felis domestici. Although domestic feline hepatozoonosis has since been recorded from Europe, Africa, Asia and America, its description, classification and pathogenesis have remained vague and the distinction between different species of Hepatozoon infecting domestic and wild carnivores has been unclear. The aim of this study was to carry out a survey on domestic feline hepatozoonosis and characterize it morphologically and genetically.A Hepatozoon sp. DNA was amplified by PCR from the blood of 55 of 152 (36%) surveyed cats in Israel and from all blood samples of an additional 19 cats detected as parasitemic by microscopy during routine hematologic examinations. Hepatozoon sp. forms were also characterized from tissues of naturally infected cats.Hepatozoon felis except for two infected by Hepatozoon canis. A significant association (p = 0.00001) was found between outdoor access and H. felis infection. H. felis meronts containing merozoites were characterized morphologically from skeletal muscles, myocardium and lungs of H. felis PCR-positive cat tissues and development from early to mature meront was described. Distinctly-shaped gamonts were observed and measured from the blood of these H. felis infected cats. Two fetuses from H. felis PCR-positive queens were positive by PCR from fetal tissue including the lung and amniotic fluid, suggesting possible transplacental transmission. Genetic analysis indicated that H. felis DNA sequences from Israeli cats clustered together with the H. felis Spain 1 and Spain 2 sequences. These cat H. felis sequences clustered separately from the feline H. canis sequences, which grouped with Israeli and foreign dog H. canis sequences. H. felis clustered distinctly from Hepatozoon spp. of other mammals. Feline hepatozoonosis caused by H. felis is mostly sub-clinical as a high proportion of the population is infected with no apparent overt clinical manifestations.DNA sequencing determined that all cats were infected with H. felis, which has predilection to infecting muscular tissues, and is highly prevalent in the cat population studied. The lack of previous comprehensively integrated data merits the redescription of this parasite elucidating its parasitological characteristics.This study aimed to integrate new histopathologic, hematologic, clinical, epidemiological and genetic findings on feline hepatozoonosis and promote the understanding of this infection. The results indicate that feline infection is primarily caused by a morphologically and genetically distinct species,  Hepatozoon species are apicomplexan parasites with a hematophagous arthropod final host and a vertebrate intermediate host. They are transmitted by ingestion of the final host containing mature oocysts by the intermediate host  . The DNHepatozoon parasites found in cats from this study was carried out using the HAM-1 F and HPF-2R primers which amplified a 1400 bp fragment of the H. felis 18S rRNA gene. Amplification of this large fragment of the 18S rRNA gene was carried out for selected samples included in the phylogenetic analyses. In addition to the DNA sequences from cats, fragments of H. canis from local Israeli dogs were also amplified using the two sets of primers and sequenced in order to compare H. felis and H. canis from cats with a local H. canis from dogs. Details on Hepatozoon sequences from this study submitted to GenBank are included in Table Further investigation of the genetic identity of 18S rRNA fragment amplified by the Piroplasmid primers , whereas the H. canis sequences found in 2 cats from the study clustered together with H. canis from dogs. Similar clustering was obtained also with the Maximum-Likelihood and the Minimum-Evolution algorithms. A Maximum Likelihood tree based on 970 bp from the longer 18S rRNA fragment amplified by primers HAM-1 F and HPF-2R (Figure H. felis sequences from Israeli cats clustered together with the H. felis Spain1 and Spain 2 sequences. Likewise, H. canis from 2 Israeli cats clustered together with H. canis [GenBank:AY461378] from a dog in Spain. These were separated from H. americanum and Hepatozoon spp. reported from other animal hosts.A Neighbor Joining phylogenetic tree based on 345 bp from the shorter H. felis by PCR were visualized by microscopy and measured (Table H. canis gamonts from dog blood, gamonts of H. felis were different due to their generally round nucleus, which is dissimilar to the more elongated horse-shoe shaped H. canis nucleus. They were also relatively shorter with a mean length of 10.5 μm compared with the H. canis gamont which is 11 μm long [H. canis within its canine counterpart. The H. felis gamont was often hardly apparent within the host cell cytoplasm and almost concealed by its nucleus.The life stages visible in the blood and tissues of cats positive for ed Table . Gamontsed Table . Some ga μm long  and lessH. felis meront is round to oval with a mean length of 39 μm by 34.5 μm and surrounded by a thick membrane separating it from the surrounding tissue (Table H. felis meronts contain amorphous material without obvious zoites (Figure H. felis merozoites are dispersed within the meront without an obvious pattern of arrangement. Early H. felis meronts appear to have thinner capsules that widen and become thicker as the meront matures. No apparent inflammatory response was found associated with the presence of intact meronts in muscular tissues (Figure The ue Table . The ears Figure  and 7. Ts Figure .H. felis meront is larger in size than the H. canis meront which is 30.6x 28.9 μm [H. canis which infects hemolymphoid and parenchymal tissues, but not muscles. Furthermore, the H. felis meront does not form the typical wheel spoke shape of the H. canis meront with merozoites arranged in a circle along the meront circumference around a central core [The  28.9 μm , found iral core .Hepatozoon muris was described from a laboratory rat and its life cycle described [H. felis, which has predilection to infecting muscular tissues, and is highly prevalent in the cat population studied. The lack of previous comprehensively integrated data merits the redescription of this parasite elucidating its parasitological characteristics.Although hepatozoonosis of domestic cats was initially reported in 1908, the same year when the type species escribed , it has Hepatozoon sp. infection in domestic cats describe variable rates of infection in different areas. A study of myocardium specimens from 100 cats brought to necropsy in Israel found that 36% of cats harbored cardiac Hepatozoon-like meronts [18S rDNA sequences from cats and dogs were closest to H. canis[H. canis was the cause of both canine and feline infections. However, the genetic analyses in this study were probably made before the sequences designated as H. felis were deposited in GenBank [Hepatozoon sp. which clustered with H. felis on a phylogenetic analysis [Surveys of  meronts . Interes meronts , 16% in  meronts , and 4%  meronts . A compa H. canis. These fanalysis .Hepatozoon spp. [H. felis. Other Hepatozoon spp. have been demonstrated to be transmitted by fleas, ticks, mites, lice, mosquitoes and sandflies [H. felis would also be transmitted by an hematophagous arthropod. The existence of more than one route of transmission for H. felis is also optional as other Hepatozoon spp. have been shown to be transmitted both by arthropod vectors and by additional routes, for instance H. canis is transmitted by the tick Rhipicephalus sanguineus as well as transplacentally [H. americanum is transmitted by the tick Amblyomma maculatum and by carnivorism [The lack of association between age and infection found in the study supports the possibility of transplacental transmission and kittens being born already infected. It may also suggest intensive exposure at a young age. The highly significant association found between infection and access to outdoors suggests the possibility of transmission by arthropod vectors, such as fleas, ticks or mites, which are common ectoparasites of cats globally, or by carnivorism as described for a number of oon spp. -5. So faandflies  and it icentally ,29, and nivorism .Hepatozoon sp. meronts, had evident gamonts [Hepatozoon infection, this could be due to the small number of samples included, as proportionally, more FIV+ cats were infected with Hepatozoon than FIV negative cats. FIV or FeLV infections have also been described in conjunction with feline hepatozoonosis in other studies [Hepatozoon infection may escape control by the immune-system in immune-suppressed cats, allowing the intensification of parasitemia and increasing the likelihood of detection by blood smear microscopy [The level of parasitaemia is usually low in feline hepatozoonosis with less than 1% of the neutrophils and monocytes containing gamonts . A surve gamonts . This st gamonts . Feline  gamonts . Althoug studies ,14. It icroscopy ,11. The croscopy .Hepatozoon sp. meronts have been reported in the muscles of domestic cats with hepatozoonosis [H. felis which infects myocardial and skeletal cat muscles, and not another Hepatozoon sp. However, other tissues such as the lungs were also infected with meronts, and PCR detected the presence of the parasite’s DNA in hemolymphoid organs such as the spleen, bone marrow and lymph nodes, and in the liver, pancreas, and kidney, as well as in fetal lungs and amniotic fluid. No substantial inflammatory response surrounding meronts was seen in the muscles examined in this study, as well in the other reports [Hepatozoon sp. has also been reported in wildlife felids and carnivores where H. felis or closely related species are responsible for myositis and myocarditis [Hepatozoon americanum infection of dogs and wildlife in the USA also has a predilection to muscle tissue, like H. felis, differing substantially from H. canis in dogs, which is found mostly in the hemolymphoid tissues and does not directly infect muscle. However, H. americanum induces the formation of muscle cysts which are much larger than H. felis meronts, are composed of concentric layers of muco-polysaccharide material surrounding a core zoite in a formation described as “onion skin” cysts, and elicit severe and painful pyogranulomatous myositis following merogony [Feline hepatozoonosis is associated mostly with infection of muscle tissues. zoonosis ,11, and zoonosis . The gen reports ,11. Thiscarditis -35. Hepamerogony ,36,37.18S rRNA Hepatozoon sequences amplified from domestic cats in this study revealed that both H. felis and H. canis infect cats in Israel, although H. felis is by far more common. Israeli H. felis sequences are indistinguishable from those reported from Spain and Brazil, and closely related to those reported in wildlife felids and carnivores from India, Korea, Japan, Tanzania, Brazil and Argentina [H. felis clusters away from H. canis and other Hepatozoon species, and is responsible for infection of domestic cats and likely also of other carnivores.The phylogenetic placement of rgentina -35,38-40H. felis is the predominant species of Hepatozoon that infects domestic cats and wild felids globally. Its wide geographic distribution could be due to transmission by some ubiquitous vector such as a common flea, mite or tick species, or to highly successful alternative routes of transmission such as transplacental transmission or carnivorism of a yet unknown wildlife intermediate host. Hepatozoon canis has been shown to spread rapidly in a young dog shelter population, and it is possible that H. felis may also spread rapidly under similar conditions [It is most likely that nditions .H. felis from multiple tissues and blood of the same cats, in conjunction with a broad-based epidemiological study, facilitated detailed characterization and redescription of this species in its intermediate host, the domestic cat. Further studies are needed to elucidate its definitive host, likely an arthropod vector, and other transmission pathways with transplacental transmission as a probable option.Merging of morphologic and genetic findings on The authors declare that they have no competing interests.GB designed the study, analyzed data wrote the manuscript; AS collected samples, performed PCR and analyzed data; SH performed and analyzed the histopathology; JPB contributed samples and assisted in writing the manuscript; YA analyzed collected samples and blood smears; DTE performed PCR on histopathological specimens, performed genetic analyzes and assisted in writing the manuscript. All authors read and approved the final version of the manuscript.


Nodal/activin signaling plays a key role in anterior-posterior (A-P) axis formation by inducing the anterior visceral endoderm (AVE), the extraembryonic signaling center that initiates anterior patterning in the embryo. Here we provide direct evidence that the mitogen-activated protein kinase (MAPK) p38 regulates AVE specification through a crosstalk with the Nodal/activin signaling pathway. We show that p38 activation is directly stimulated by Nodal/activin and fails to be maintained upon inhibition of this pathway both in vivo and in vitro. In turn, p38 strengthens the Nodal signaling response by phosphorylating the Smad2 linker region and enhancing the level of Smad2 activation. Furthermore, we demonstrate that this p38 amplification loop is essential for correct specification of the AVE in two ways: first, by showing that inhibiting p38 activity in 5.5 days postcoitum embryo cultures leads to a switch from AVE to an extraembryonic visceral endoderm cell identity, and second, by demonstrating that genetically reducing p38 activity in a Nodal-sensitive background leads to a failure of AVE specification in vivo. Collectively, our results reveal a novel role for p38 in regulating the threshold of Nodal signaling and propose a new mechanism by which A-P axis development can be reinforced during early embryogenesis. ► MAPK p38 signaling is essential for specification of the A-P axis in the mouse embryo ► Activation of p38 is mediated by Nodal signaling prior to gastrulation ► Phosphorylation of the Smad2 linker region by p38 enhances Smad2 activation ► Nodal signaling requires p38 amplification to induce the anterior visceral endoderm The anterior-posterior (A-P) axis of the mammalian embryo is the first of the definitive embryonic axes to be determined. The A-P axis is initiated by the induction of the anterior visceral endoderm (AVE) at the distal tip of the 5.5 days postcoitum (dpc) embryo and its migration to the prospective anterior of the embryo shortly after . Nodal sHex-GFP [Lim1, Hex, Dkk1, and Lefty1/2 was completely lost , and the expression of mesoderm patterning markers Eomes, Fgf8, T, and Snail was not diminished when 6.5 dpc embryos were cultured overnight in the presence of the p38 inhibitor  form of p38 (p-p38) was highest in the cytoplasm of visceral endoderm cells, with some of these cells also showing nuclear localization. Weak expression was also observed in the cytoplasm of epiblast cells at these stages A and 2B.Nodal mutant embryos, p38 phosphorylation was severely downregulated or lost in both Nodal and Cripto mutants at 5.5 dpc  cells, which are primitive endoderm stem cells and provide an in vitro model for extraembryonic endoderm development . XEN celWe next set out to establish how p38 was affecting AVE gene expression. We asked whether p38 inhibition affected the activation of Smad2, the effector of canonical Nodal/activin signaling . XEN celTo analyze whether p38 inhibition also affected Smad2 target gene expression, we analyzed how p38 inhibition affected stimulation by activin of the Smad2 responsive element SBE4 . This reDkk1 and Lim1 was completely abolished. In contrast to this, in embryos treated with the p38 inhibitor plus activin there was a partial rescue of the expression of these genes. In p38 inhibitor plus activin-treated embryos, although the expression Dkk1 and Lim1 was weaker than in controls and had not migrated to the prospective anterior, the expression of these genes could be clearly observed at the distal tip of the embryo . We anaThe fact that p38 is working independently of the termination of p-Smad2 signaling suggests that it must be acting upstream of these events. For this reason we analyzed whether p38 is affecting the rate of Smad2 activation by carrying out a time course of Smad2 activation kinetics in response to activin treatment and analyzing how this is affected by p38 inhibition. We observed that phosphorylation of Smad2 is stimulated as early as 5 min after activin treatment and that even at this early time point, p38 inhibition reduces the overall level of C-terminal p-Smad2 C. This iWe next tested what the impact of phosphorylation of the Smad2 linker region by p38 MAPK has on the Smad2 signaling activation. For this we used a Flag-tagged Smad2 construct where the MAPK linker region phosphorylation sites have been mutated (Flag-Smad2EPSM) . A critiLim1 at 5.5 dpc and Lim1, Cerl1, and Lefty1 at 6.5 dpc, in either control embryos or p38IP−/− embryos, we observed that all three genes are appropriately expressed , showing that p38 and Nodal cooperate during AVE specification. These results indicate that p38 activity is required to enhance Nodal signaling in the embryo and therefore that the crosstalk between p38 and Nodal signaling is essential to establish the A-P axis during mouse development.Given that we have found that p38 is critical to achieve maximal Nodal signaling, we set out to test this requirement genetically and in vivo. p38IP is a p38 interacting protein required for p38 activity and mesoderm migration during early mouse development . Little xpressed E. In conGraded Nodal signaling is central to axis specification in the vertebrate embryo, and how the different thresholds of signaling are achieved determines what cell identity is specified by this pathway. At the time that the AVE is induced, high levels of expression of extracellular and intracellular inhibitors of Nodal signaling that modulate A-P axis specification have been reported . HoweverOur data suggest that the interaction between p38 and the Nodal/activin signaling pathways occurs at least at two levels. First, the TGF-β ligands Nodal/activin as well as the coreceptor Cripto can stimulate p38 phosphorylation and therefore activate this MAPK. Second, p38 in turn enhances Nodal/activin signaling within the visceral endoderm via phosphorylation of the Smad2 linker region to ensure maximal activation of Smad2. These results contrast with those for BMP signaling where MAPK and GSK3β phosphorylation of the Smad1 linker region decreases Smad1 stability and leads to a shorter duration of BMP signaling . How do Could this interaction have any relevance to other contexts where Nodal signaling is required? During gastrulation, the posterior epiblast cells undergo an epithelial to mesenchymal transition (EMT) that is essential for proper mesoderm migration from the primitive streak. Both p38IP and p38 have been shown to be required to downregulate e-cadherin in the primitive streak and therefore are essential for proper mesoderm migration to occur . Nodal sIn conclusion, our work sheds light on how different thresholds of Nodal signaling are achieved within the embryo and on the consequences that these different thresholds have on cell fate decisions during early embryonic development. From a broader perspective, this work highlights a novel mechanism that enhances TGF-β signaling and therefore has important implications for the regulation of this pathway in development and disease.


Rice dwarf phytoreovirus (RDV), a member of Phytoreovirus that contains a 12-segmented dsRNA genome. We report here that Pns6 binds both dsRNAs and ssRNAs. Intriguingly, Pns6 exhibits non-sequence specificity for dsRNA but shows preference for ssRNA sequences derived from the conserved genomic 5′- and 3′- terminal consensus sequences of RDV. Furthermore, Pns6 exhibits magnesium-dependent ATPase activities. Mutagenesis identified the RNA binding and ATPase activity sites of Pns6 at the N- and C-termini, respectively. Our results uncovered the novel property of a viral MP in differentially recognizing dsRNA and ssRNA and establish a biochemical basis to enable further studies on the mechanisms of dsRNA viral MP functions.Cell-to-cell movement is essential for plant viruses to systemically infect host plants. Plant viruses encode movement proteins (MP) to facilitate such movement. Unlike the well-characterized MPs of DNA viruses and single-stranded RNA (ssRNA) viruses, knowledge of the functional mechanisms of MPs encoded by double-stranded RNA (dsRNA) viruses is very limited. In particular, many studied MPs of DNA and ssRNA viruses bind non-specifically ssRNAs, leading to models in which ribonucleoprotein complexes (RNPs) move from cell to cell. Thus, it will be of special interest to determine whether MPs of dsRNA viruses interact with genomic dsRNAs or their derivative sRNAs. To this end, we studied the biochemical functions of MP Pns6 of  Tobacco mosaic virus, encode MPs that can alter the size exclusion limit (SEL) of plasmodesmata Grapevine fanleaf virus, move through tubular structures formed inside modified plasmodesmata that are induced by the viral MPs Potato virus X (PVX), has RNA binding, RNA helicase and Mg2+-dependent ATPase activities in vitro Nicotiana clevelandiiCell-to-cell movement is required for both local and systemic infection by plant viruses. Viruses encode movement proteins (MP) to facilitate such movement. The specific movement mechanisms vary among viruses. Some viruses, such as Rice dwarf phytoreovirus (RDV) is a member of the genus Phytoreovirus within the family Reoviridae that replicates in both host plants and insect vectors. The genome of RDV is composed of 12 double-stranded RNAs (dsRNAs) 5′ GGCAAA--- or 5′ GGUAAA---) and a 3′ terminal consensus sequence (---UGAU 3′ or ---CGAU 3′) in vivo and is localized at the periphery of viroplasms Nicotiana tabacum bombarded and RDV-infected rice leaves. However, Pns6 did not suppress RNA silencing in cells The RDV virion has an outer shell composed of structural proteins P2, P8 and P9, and a core composed of structural proteins P1, P3, P5 and P7 as well as the genomic dsRNAs. The functions of nonstructural proteins Pns4, Pns6, Pns10, Pns11 and Pns12 have previously been characterized. Pns4 can be phosphorylated To advance our understanding of the cell-to-cell movement mechanisms of dsRNA viruses, we studied the biochemical functions of RDV Pns6. We report here that Pns6 binds both dsRNAs and ssRNAs. Intriguingly, Pns6 exhibits non-sequence specificity for dsRNA but shows preference for ssRNA sequences derived from the conserved genomic 5′- and 3′- terminal consensus sequences of RDV. Furthermore, Pns6 exhibits magnesium-dependent ATPase activities. Mutagenesis identified the Pns6 RNA binding and ATPase activity sites at the N- and C-termini, respectively. Our results uncovered the novel property of a viral MP in differentially recognizing dsRNA and ssRNA and establish a biochemical basis to enable further studies on the mechanisms of dsRNA viral MP functions.In our previous study, Pns6 was shown to restore the cell-to-cell movement activity of a PVX 25 K deletion mutant During RDV replication, both dsRNAs and ssRNAs are produced. Therefore, both forms of RNA  were useE. coli, showed no interaction with the RNAs. These controls ruled out the possibility that the observed Pns6-RNA interaction was due to (i) non-specific electrostatic interactions or (ii) contaminating proteins from E. coli that bound the RNAs. Rather, they indicate that Pns6 interacted with the RNAs via specific molecular recognition. This specificity is further supported by the following experimental results showing selectivity of Pns6 for some RNA sequences and identifying the Pns6 domain for RNA binding.Many MP-RNA complexes were disrupted at high salt concentrations Genomic RNAs of RDV consist of 12 dsRNA segments containing consensus sequences at their 5′ and 3′ ends. As described above, Pns6 showed stronger binding to ssRNA probes containing these conserved sequences than to a random ssRNA . We condTo test sequence preference of ssRNA for Pns6 binding, three ssRNA competitors with random sequences and different lengths  were used to compete with a conserved ssRNA sequence (S3-5a). The richness of basic amino acids in the N-terminal region of Pns6 suggests a role of this region in RNA binding. To test this, we generated a series of Pns6 mutants to test for RNA binding. As shown in E. coli. It was previously reported that divalent cations play an important role in ATP hydrolysis reactions The putative GKS motif at amino acid positions 125 to 127 suggests that Pns6 may be an ATPase. We performed a thin layer chromatography (TLC) assay with purified His-tagged Pns6. We performed TLC and colorimetric malachite green assays with purified mutant Pns6 proteins to locate the potential ATPase activity site. Mutants M1, M3, M13 and M23 showed ATPase activity equal to or higher than that of wild-type Pns6 in TLC assays . MutantsAlthough our knowledge of the detailed RDV replication cycle is still very limited, it has been proposed that the this virus may have a similar lifecycle to animal reoviruses Virions or viral ribonucleoprotein complexes (vRNPs) can be transported through plasmodesmata into adjacent cells. In the case of RDV, it is unclear whether the virus is transported in the form of virions or vRNPs. RDV virions spread among cells of insect vectors through tubular structures composed of RDV Pns10 Pns6 binds dsRNA in a non-sequence-specific manner. Many known MPs, such as the 30 K protein of TMV, bind ssRNA cooperatively and sequence-non-specifically The ATPase activity of Pns6 may be important for RDV movement. Numerous plant virus MPs have ATPase activities In summary, our analyses of RDV Pns6 uncovered the novel property of a viral MP in differentially recognizing dsRNA and ssRNA. Such property, together with the identification of the RNA binding sites and ATPase activity site in the protein, establishes a biochemical basis to enable further studies on the mechanisms of dsRNA viral MP functions in movement and perhaps also other aspects of the viral life cycle.BamH I and Sal I, and the resulting S6/S7/S9 segments were inserted into the prokaryotic expression vector pET28a to yield pET28a-S6, pET28a-S7, pET28a-S9. Primers for constructing the S6 mutant segments are listed in EcoR V site to generate pBS-mutant plasmids. After digesting the pBS-mutant plasmids with BamH I, the resulting mutant fragments were ligated into the prokaryotic expression vector pGEX-4T-1 at the BamH I site to generate pGEX-mutant plasmids. The BamH I/Xho I fragment released from the pGEX-mutant was inserted into pET28a at the BamH I/Xho I site to produce pET28a-mutant plasmids.Plasmids pGEM-S6, pGEM-S7 and pGEM-S9 were digested with Escherichia coli BL21 cells (TaKaRa) containing pET28a-S6, pET28a-S7, pET28a-S9 or pET28a-S6 mutant were used to produce His-tagged Pns6, P7, P9 and mutant proteins, and BL21 cells carrying pGEX-mutant constructions were used to produce GST-fused S6 mutant proteins. The cells were cultured in Luria-Bertani medium until they reached an OD600 of 0.6, and then the recombinant proteins were induced by adding isopropyl β-D-thiogalactoside (0.5 mM) for 2 hours at 37°C or overnight at 18°C. The soluble recombinant proteins were purified using affinity chromatography with a Ni2+-chelating column or a Glutathione Sepharose 4B column. The His-tagged proteins were purified in a buffer containing 20 mM TrisCl and 500 mM NaCl, pH 7.0, and the GST fused proteins were purified in a buffer containing 40 mM Tris and 50 mM NaCl, pH 8.0. The resulting proteins were used for ATPase analysis and electrophoresis mobility shift assays (EMSA).Proteins in inclusion bodies were isolated and recovered for northwestern blotting analysis. Cells were harvested by centrifugation at 5,000 g at 4°C for 15 min and resuspended in lysis buffer  at 4°C. After sonication and centrifugation at 20,000 g at 4°C for 15 min, the resulting pellet was washed once with the same lysis buffer. The washed inclusion bodies were resuspended in 6 M guanidine hydrochloride, 100 mM TrisHCl pH 7.0, 100 mM dithiothreitol, 1 mM EDTA. The solubilized proteins were separated by SDS-PAGE. The gel was stained with cold 0.5 M KCl. The respective band was then cut out, crushed, and mixed with the SDS-PAGE loading buffer. After 10 min incubation at 80°C, the sample was centrifuged for 15 min at 13,000 g and the supernatant was collected for northwestern blotting.32P-ATP using T4 polynucleotide kinase (NEB).The sequences of the synthesized RNA probes and the templates used for the in vitro transcription are presented in Northwestern blotting was used to detect the nucleic acid binding activity of Pns6 and its mutants as described previously For EMSA, RNA probes were incubated with various concentrations of proteins for 20 min on ice in a 10 µl reaction system containing 50 mM NaCl, 50 mM Tris⋅HCl, pH 7.5, and 10% glycerol. Samples were separated by native PAGE in a 9% gel in 0.5×TBE buffer for 80 minutes at 180 V, and then the gel was dried for autoradiography. In the competition assays, unlabeled RNAs were added to compete with the labeled RNA probes. The competition ability of the RNAs is related to the signal from the free RNA probes, which was quantified with ImageJ (version 1.4) software.2, 1 mM DTT, 0.5 mM ATP and the respective proteins. α-32P-ATP was added to the reactions at 0.05 µCi/µl. After a 30 min incubation at 37°C, 0.5 µl of the reaction product was dotted onto a PEI thin layer chromatography plate (Merck), and the plate was developed in 0.15 M LiCl and 0.15 M formic acid as described previously In the TLC ATPase analysis, we used a 10 µl reaction system containing 50 mM Tris, 5 mM MgCl2, 1 mM DTT, 1 mM ATP and the respective protein. A 96-well microplate was used to analyze multiple samples. After 60 min of incubation at 37°C, 45 µl of the colorimetric mixture  was added to the reactions. To develop the reaction, 45 µl of 15% sodium citrate was added. The OD570 of the samples was read by a TECAN SUNRISE basic scanner.The colorimetric malachite green assay was performed as described previously


To investigate whether uric acid (UA) is an independent predictor of cardiovascular (CV) and all-cause mortality in peritoneal dialysis (PD) patients after controlling for recognized CV risk factors.A total of 2264 patients on chronic PD were collected from seven centers affiliated with the Socioeconomic Status on the Outcome of Peritoneal Dialysis (SSOP) Study. All demographic and laboratory data were recorded at baseline. Multivariate Cox regression was used to calculate the hazard ratio (HR) of CV and all-cause mortality with adjustments for recognized traditional and uremia-related CV factors.There were no significant differences in baseline characteristics between patients with (n = 2193) and without (n = 71) UA measured. Each 1 mg/dL of increase in UA was associated with higher all-cause mortality with 1.05(1.00∼1.10) of HR and higher CV mortality with 1.12 (1.05∼1.20) of HR after adjusting for age, gender and center size. The highest gender-specific tertile of UA predicted higher all-cause mortality with 1.23(1.00∼1.52) of HR and higher CV mortality with 1.69 (1.21∼2.38) of HR after adjusting for age, gender and center size. The predictive value of UA was stronger in patients younger than 65 years without CV disease or diabetes at baseline. The prognostic value of UA as both continuous and categorical variable weakened or disappeared after further adjusted for uremia-related and traditional CV risk factors.The prognostic value of UA in CV and all-cause mortality was weak in PD patients generally, which was confounded by uremia-related and traditional CV risk factors. Increased cardiovascular (CV) events have been extensively documented in patients with end-stage renal disease (ESRD) including peritoneal dialysis (PD) and hemodialysis(HD) population Uric acid (UA), as one of novel risk factors, has been paid more attention in recent years. In general population, previous studies have shown that UA is closely associated with hypertension, coronary heart disease and chronic kidney disease (CKD) In the present study, we aimed to explore associations of UA, all-cause and CV mortality in a large-scale multi-center PD cohort. The prognostic value of UA would be compared between patients≥65 years and <65years, with or without CV disease (CVD), diabetes and non-diabetes at baseline respectively. In addition, we determined whether associations of UA and outcomes would be changed after controlling for uremic-related factors and traditional CV risk factors .This is an affiliated study with the Socioeconomic Status on the Outcome of Peritoneal Dialysis (SSOP) Study, which is a retrospective multi-center cohort study as described in detail in our previous paper Centers which have professional PD doctors and PD nurses, and have well-developed databases of at least 3-years duration, recording baseline characteristics and follow-up data every 1∼3 months for each patient in our country participated this study voluntarily. Totally 9 centers were qualified, and 7 of them agreed to participate providing about 70 percent of all incident patients from 9 centers. Enrolled centers were from 5 provinces and located at 4 geographical regions  in China. Data from each center have been collected within the strict quality control framework and further inspected and optimized to keep integrity and accuracy of the database. All study investigators and staff members completed a training program that taught them the methods and processes of the study. A manual of detailed instructions for data collection was distributed.All the incident patients on chronic PD between the date of intact database creation and August 2011 were enrolled into this study. Each patient signed informed consent to agree their demographic and lab data to be used in future studies since they started PD therapy. All subjects began the PD program within one month after catheter implantation and were given lactate-buffered glucose dialysate with a twin-bag connection system .Demographic and clinical data including age, gender, body mass index (BMI), socioeconomic status , primary renal disease, the presence of cardiovascular disease (CVD) and diabetes mellitus (DM) were collected at baseline. Center size was also recorded according to number of enrolled patients of each center. CVD was recorded if one of the following conditions was present: angina, class III–IV congestive heart failure (NYHA), trandient ischemic attack, history of myocardial infarction or cerebrovascular accident and peripheral arterial disease Blood pressures were measured according to the guidelines presented in the Seventh Report of the Joint National Committee on Prevention, Detection, Evaluation and Treatment of High Blood pressure Biochemistry data including hemoglobin, serum albumin, UA, lipids spectrum, glucose, calcium, phosphate and intact parathyroid hormone (iPTH) were examined using an automatic Hitachi chemistry analyzer. The first testing was completed within one month of PD at the first visit, and then repeatedly once a month. The mean values in the first 3 months were calculated. The coefficient of variation of UA from multiple measurements was 5.3% for subjects from Peking University First Hospital but not recorded for those from other hospitals. Serum UA was measured by the uricase method using the same autoanalyzer. Serum high sensitive C-reactive protein (CRP) was measured by immune rate nephelometric analysis. Dialysis adequacy and residual renal function (RRF) were measured after one month of dialysis therapy. RRF was defined as the mean of residual creatinine clearance and residual urea clearance. Dialysis adequacy was defined as total Kt/V and total creatinine clearance. Corrected calcium was calculated by standard equation: Corrected calcium = serum total calcium+0.02*.The outcomes were defined as cardiovascular and all-cause death. The cardiovascular death was defined as death due to myocardial infarction, congestive heart failure, cerebral bleeding, cerebral infarction, arrhythmia, peripheral arterial disease, and sudden death. In all analysis, we censored follow-up at trandferring to HD, loss to follow-up, renal trandplantation, or the end of the study .Continuous data were presented as mean with SDs except for CRP and RRF, which were presented as median (interquartile range) because of a high skew. Categorical variables were presented as proportions. Patients' data were compared by using the t-test or ANOVA F-test for normally distributed continuous variables, chi-square test for categorical variables, and Mann-Whitney U test for skewed continuous variables. UA was trandformed into categorical variable by gender-specific tertiles or quartiles.For determining associations of UA, CV and all-cause mortality, UA as continuous variable was first examined in Cox regression models after adjusting for age, gender and center size (model 1) for all participants, and then in subgroups such as patients≥65 years or <65 years, with or without CVD, with or without DM respectively. Next, we explored whether associations of UA and CV/all-cause mortality in all participants were confounded by traditional and uremia-related CVD factors. Uremia-related factors such as serum albumin, hemoglobin, phosphate, RRF and CRP (model 2), and additional traditional CV factors such as BMI, the history of diabetes or CVD, mean arterial pressure and LDL cholesterol(model 3) were constructed respectively. For these examinations, UA was also considered as categorical variable by gender-specific tertiles or quartiles respectively but only gender-specific tertiles of UA was shown in the context since similar linear trends were indicated. Gender was not included as the adjusted variable if UA is examined as the gender-specific variable.We reported the multivariable adjusted hazards ratios (HRs) with 95% CIs. All probabilities were two-tailed, and the level of significance was set at 0.05. Statistical analysis was performed by SPSS for Windows software version 13.0 .2; 37.7% were diabetic and CVD was present in 41.5% of subjects at baseline. Chronic glomerulonephritis was the most common cause of ESRD (34.4%), followed by diabetic nephropathy (29.3%) and hypertensive nephropathy (15.5%). There were 71 of 2264 patients without UA values at baseline. The mean age, BMI, MBP, serum lipids, distribution of primary renal disease, CVD history, prevalence of DM were not significantly different between those who had measured UA and those who did not (2193 patients) (P>0.05). A total of 80 patients with inactive solid organ tumors at baseline were not excluded since their UA values were comparable to the remainders, and linear trends of UA and outcomes were not changed when they were excluded. Thereafter, 2193 patients were included in the final analysis.A total of 2264 PD patients were collected, with a mean age of 58.1±15.5 years, BMI of 22.9±3.6 kg/mThe median follow-up time was 26.5(13.6∼43.6) months. At the end of study, of 586 (26.7% of 2193) patients who died, 231 cases (39.4%) were due to CVD, 140 cases (23.8%) were due to infection, and other causes of death included malignancy (11.9%), gastrointestinal bleeding (4.3%), severe malnutrition (4.8%), miscellaneous (5.1%), and undefined (10.6%). Of 231 patients who died from CVD, the leading cause was myocardial infarction , followed by congestive heart failure (19.0%), cerebral bleeding (14.3%), cerebral infarction (12.9%), sudden death (9.5%), arrhythmia (4.8%), peripheral arterial disease (1.3%), and undefined causes (15.1%).P>0.05).The mean values of UA were 6.41±1.87 mg/dL for the whole cohort. The normal distribution of UA in male and female was shown respectively in P = 0.05) and higher CV mortality with 1.12 (1.05∼1.20) of HR(P = 0.001) after adjusting for age, gender and center size ( and ). We further divided patients into subgroups, i.e.age≥65 years and <65years, with and without CVD, DM and non-DM respectively. For CV mortality rather than all-cause mortality, the prognostic value of UA was significant in low-risk groups such as patients with age<65 years, without CVD or DM at baseline rather than in their counterpars respectively ( and P = 0.04) and higher CV mortality with 1.69 (1.21∼2.38) of HR(P = 0.002) after adjusting for age, gender and center size compared to low tertile of UA. However, the associations of UA and CVD/all-cause mortality weakened after further adjusted for uremia-related factors including serum albumin, hemoglobin, phosphate, and CRP, and disappeared with additional adjustement for traditional CV factors such as CVD history, DM, body mass index, and LDL cholestrol ( and ).The associations between UA and outcomes were analyzed. First, UA was examined as a continuous variable. Each 1 mg/dL of increase in UA was associated with higher all-cause mortality with 1.05(1.00∼1.10) of HR.In conclusion, we suggested that high UA is weakly associated with CV and all-cause mortality for PD population. More large-scale PD cohort studies are needed to verify our findings. Whether high UA levels should be modified for PD patients as done for CKD patients is to be determined.


Ulcerative colitis (UC) was the most frequently diagnosed inflammatory bowel disease (IBD) and closely linked to colorectal carcinogenesis. By far, the underlying mechanisms associated with the disease are still unclear. With the increasing accumulation of microarray gene expression profiles, it is profitable to gain a systematic perspective based on gene regulatory networks to better elucidate the roles of genes associated with disorders. However, a major challenge for microarray data analysis is the integration of multiple-studies generated by different groups.In this study, firstly, we modeled a signaling regulatory network associated with colorectal cancer (CRC) initiation via integration of cross-study microarray expression data sets using Empirical Bayes (EB) algorithm. Secondly, a manually curated human cancer signaling map was established via comprehensive retrieval of the publicly available repositories. Finally, the co-differently-expressed genes were manually curated to portray the layered signaling regulatory networks.Overall, the remodeled signaling regulatory networks were separated into four major layers including extracellular, membrane, cytoplasm and nucleus, which led to the identification of five core biological processes and four signaling pathways associated with colorectal carcinogenesis. As a result, our biological interpretation highlighted the importance of EGF/EGFR signaling pathway, EPO signaling pathway, T cell signal transduction and members of the BCR signaling pathway, which were responsible for the malignant transition of CRC from the benign UC to the aggressive one.The present study illustrated a standardized normalization approach for cross-study microarray expression data sets. Our model for signaling networks construction was based on the experimentally-supported interaction and microarray co-expression modeling. Pathway-based signaling regulatory networks analysis sketched a directive insight into colorectal carcinogenesis, which was of significant importance to monitor disease progression and improve therapeutic interventions. TP53, K-ras, APC, Wnt5, beta-catenin, DCC or microRNAs APC and DCC were the most frequently detectable prognostic signatures for CRC; however, the results were perplexed and cardinal hurdles for clinical therapeutic interventions were still insurmountable. Since most of variant genes have ineffectual profits for diagnosis and underlying mechanisms associated with CRC are ill-defined.As the fourth commonest carcinoma, colorectal cancer (CRC) associated with significant cause of mortality worldwide owing to its prevailing distant metastasis Considerable documents certified countless malignancies occurred in association with chronic inflammation. Infection with hepatitis B or C viruses had been found to be the main cause of hepatocellular carcinoma cis-elements, and regulators. The regulators, also called transcription factors, are often described as proteins which bound to specific regions of target gene, and thereby regulated the transcription of genetic information from DNA to mRNA cis-elements in GRNs and thereby control the level of gene expression during transcription.Cells in multi-cellular organisms switch into diverse fates, such as division, proliferation, apoptosis or differentiation into specialized phenotypes. Genome-wide association studies (GWAS) of gene regulatory networks (GRNs) govern this process and the inference of GRNs is crucial for understanding underlying molecular mechanisms between genes and gene regulation As yet, a wide variety of approaches have been proposed for modeling GRNs, such as discrete models of Boolean networks However, mining these data to better comprehend gene expression and regulation proposes a major challenge for bioinformatics. Since practical considerations restrain the size of samples and the overlap genes in multiple studies are limited with poor predictability The purpose of functional genomics in the post-genome era is to better elucidate molecular mechanisms involved in gene regulation et al. network, a comprehensive analysis of human cancer signaling architectural organization assembled from cancer-associated genetically and epigenetically altered genes was established Traditionally, cell signaling is described as linear diagrams. As more cross-talk between signaling has been reviewed, a systematic network view of cell signaling is proposed In this study, we modeled a layered signaling regulatory network associated with colorectal cancer from cross-study microarray gene expression data using the experimentally-supported interaction and microarray co-expression modeling. Cytoscape http://www.ebi.ac.uk/arrayexpress/), Gene Expression Omnibus  and Stanford Microarray Database  for microarray data sets satisfying the following criteria: (1) deriving from Homo sapiens; (2) depicting genome-wide co-expression information of ulcerative colitis and neoplastic lesions; (3) supplying raw data files. After extensively retrieval, two different microarray expression cohorts performed on Affymetrix GeneChip platform were downloaded from GEO and selected for further investigation  was performed on Affymetrix Human Genome U133 Plus 2.0 Array platform The first data set obtained from Gyorffy et al.The other one is a subset of 15 Affymetrix HT HG-U133 GeneChips originated from a gene expression experiment by Pekow http://www.arraymining.net/), which pooled multiple microarray data from independent investigations or platforms into a combinational cohort To standardize the microarray data sets obtained from independent studies and reduce systematic distortions produced by different laboratories, rank normalization was introduced to effectuate robust multi-array average (RMA) analysis http://www-stat.stanford.edu/~tibs/SAM/) to produce a cluster of up- or down-regulated variant genes via comparison of UC with CRC We simulated 8 virtual cohorts, called the random microarray data sets (RDSs), from the integrated data set mentioned above et al., which united manually curated signaling molecules including BioCarta (http://www.biocarta.com/) http://plasmid.hms.harvard.edu/) and Online Mendelian Inheritance in Man . Subsequently, Human Protein Reference Database  http://www.reactome.org/) databases were appended. After adding connections between signaling molecules based on SysBiomics platform (http://biomine.cigb.edu.cu/sysbiomics/) http://www.cytoscape.org/) To provide a high-quality human cancer signaling atlas with great superiority, the manually curated molecules of human cancer signaling were assembled as previously described http://www.hprd.org/), Human Proteinpedia (http://www.humanproteinpedia.org/) and EntrezGene (http://www.ncbi.nlm.nih.gov/gene/). Cerebral generated a view of the layered map afterwards and each of the signaling regulatory networks was divided into four layers, including extracellular, membrane, cytoplasm and nucleus Subcellular localization information involved in the signaling regulatory networks driven by the co-differently-expressed genes was diffusely retrieved from Human Protein Reference Database  was freely employed to enrich the potential biological functions based on “GOTERM_BP_FAT” option http://toppcluster.cchmc.org/) was selected as gene sets category Functional enrichment analysis was performed to evaluate underlying biological functions involved in the layered signaling regulatory networks. In this study, the DAVID functional annotation clustering tool (http://www.r-project.org/) cooperated with stats package was introduced to hierarchically trace heatmap for the over-represented GO biological processes and signaling pathways in the layered signaling regulatory network.To obtain an intuitionistic two-dimensional graphical representation of large-scale cluster members across an activity profile and define the main clusters by visual inspection of the resulting tree, R . Meanwhile, a heatmap with hierarchically clustering was produced to visualize these processes based on stats package in R project. As shown in To gain a detailed insight into the functions of the whole regulators in the layered signaling regulatory network, we additionally performed pathway enrichment analysis using ToppCluster. As appended in As microarray data on gene expression programs become available, it is profitable to create a systematic view of biological systems to improve our understanding of underlying mechanisms associated with disorders et al. indicated the ‘one-step-clustering’ of gene expression profiles and network-based gene signatures used in the past decade was far from sufficient to produce robust gene signatures Despite massive amounts of experimentally-supported microarray expression data bestowed in public repositories, it was still strenuous to avail legitimately. Since experiments were run on different laboratories, the combined usage of multiple platforms was necessary to overcome technical variation of individual study that resulted from sample preparation, labeling, hybridization, handling and other processing steps In this study, we modeled a layered signaling regulatory network via integration cross-laboratories microarray expression data based on the experimentally-supported interaction and microarray co-expression modeling. Pathway-based signaling regulatory networks analysis revealed EGF/EGFR signaling pathway, EPO signaling pathway, T cell signal transduction and members of the BCR signaling pathway were involved in colorectal carcinogenesis. Our biological interpretations are as follows:et al. also discovered that EGF could significantly reduce colon ulcer and inflammation in vivo after intracavitary application, suggesting EGF was a protective cytokine in ulcerative colitis Numerous experimental and epidemiological investigations supplied strong evidences that EGF/EGFR signaling pathway played a pivotal role in ulcer repair. As a single-chain polypeptide of 53 amino acid residues, epidermal growth factor (EGF) could stimulate cell growth, proliferation and differentiation through autocrine, paracrine and endocrine mechanisms. It had been indicated that EGF could accelerate ulcer repair in the experimental colitis animal model KRAS/BRAF and PI3K/AKT were certified to entangle in colorectal cancer PI3K/AKT pathway which subsequently gave rise to solid tumor proliferation. Therefore, tactics targeting EGF/EGFR cascade system would have been an effective and timely approach for clinical prevention and therapeutic intervention for the transition from chronic non-resolving UC to pernicious colorectal carcinoma However, the over-expression of EGF was an aggressive biological behavior, as increased levels of EGF/EGFR were detected in innumerable types of carcinomas In vitro and in vivo models indicating administration erythropoietin could drastically reverse the suppression related to pro-inflammatory cytokines of cellular maturation of the erythroid lineage et al. discovered recombinant human erythropoietin (rhEPO) treatment remarkably attenuated hyperoxia-induced lung injury by down-regulating inflammatory responses in neonatal rat model Pathway-based approach for analysis of GWAS also proposed the assumption that the dysfunction of EPO signaling pathway was assigned to be a highly aggressive biological process associated with colorectal carcinogenesis. Erythropoietin (EPO) was a primary growth factor regulating erythroid progenitor proliferation and maturation et al. examined the expression of the EPO/EPOR in 24 kinds of malignant cancerous cell lines and confirmed EPO signal transduction system was engaged in tumorigenesis of almost all malignancies The roles of EPO had been well documented in erythrocytopoiesis, but its clinical relevance in carcinoma remained controversial and deserved for further investigations. Activation of EPO signal transduction pathway was significantly dysregulated during disorders progression to a more aggressive phenotype. As early as 2003, EPO was suggested as a rewarding guideline for cancer patients. However, owing to an observed higher mortality, concerns were raised that EPO was unprofitable for survival in cancer patients + regulatory T cells (Treg) specifically stimulated by IL-23 is especially relevant to tissue inflammation + Treg constituted 60–80% of the whole T cells in mice with UC et al. also discovered that CD4/CD45RBhigh Treg could be confirmed as an initiator of IBD, suggesting T cell signal transduction was highly associated with chronic IBD Imbalance of anti-inflammatory mediators  and excessive pro-inflammatory responses appeared to be a risk factor for chronic IBD +CD25+ Treg, suggesting T cell signal transduction played a role in immunosuppressive effect. Meanwhile, activated suppression by Treg had been confirmed to play an important role in T cells down-regulation Several studies had shown a huge accumulation of Treg distributed in solid tumors and increased with the transition from benign to a malignant state et al. uncovered, owing to the damage involved in intestinal digestive tract, polyclonal activators could mediate local immune response through penetrating lymphoid tissues B cell receptor (BCR), a multi-protein complex with an antigen binding subunit and a signaling subunit, was crucial for inflammatory signaling initiation and propagation. With the assistance of costimulatory signals such as natural killer (NK) or M cytokines, B cell superantigen (BSAg) could interact with B cells and Ig, and participate in immune system or inflammatory disorders via cross-linking with BCR + Treg into FoxP3+Treg via direct interaction with transforming growth factor-beta (TGF-beta) pathways, resulting in tumor proliferation. However, in the absence of Breg, the transformation of FoxP3+Treg was impeded and inhibited breast cancer metastasis Aberrant BCR signaling pathway plays significant roles in the pathogenesis of tumor immunity. Recent studies demonstrated that over-expression of regulatory B cell (Breg) could enhance tumor immunity A layered signaling regulatory network involved in the transition from primary UC to aggressive CRC was successfully modeled via linking cross-study microarray co-expression models with experimentally-supported interactions from the map of human cancer signaling, which led to identifying several chief cellular processes and signaling pathways. These pathological processes had been documented to be characteristics for colorectal carcinogenesis, and could be summarized into four main pathways, including EGF/EGFR signaling pathway, EPO signaling pathway, T cell signal transduction and members of the BCR signaling pathway. Therefore, our biological interpretation was focused on these pathways and their potential contributions to the transition from benign UC to aggressive or malignant CRC.Our model for inferring the layered signaling regulatory network has two key points. For one thing, the connections between nodes or hubs in the signaling regulatory networks were integrated from cross-laboratories microarray expression profiling. For another, the layered signaling regulatory network, as opposed to the traditional ones, provided infrequent insights into molecular function and the intricate signaling pathways at the subcellular level. However, due to its positive connections and some noises that could not be avoided, the layered signaling regulatory network presented here is still not comprehensive. Nevertheless, we still confirmed that our inference would provide incentive illustration for the malignant transition of CRC, and supplied directive significance for future therapeutic intervention.Appendix S1Robust multi-array average (RMA) analysis result of GSE37283 based upon WebArray platform.(ZIP)Click here for additional data file.Appendix S2Robust multi-array average (RMA) analysis result of GSE4183 based upon WebArray platform.(ZIP)Click here for additional data file.Appendix S3Cross-study normalization and integration results (part 1) using ArrayMining.(ZIP)Click here for additional data file.Appendix S4Cross-study normalization and integration results (part 2) using ArrayMining.(ZIP)Click here for additional data file.Appendix S5Detailed list of the differently expressed genes by Significant Analysis of Microarray (SAM) originated from the simulated microarray data sets or virtual random microarray data sets (RDSs).(ZIP)Click here for additional data file.Appendix S6A map of human cancer signaling network.(ZIP)Click here for additional data file.


Uric Acid (UA), historically considered as a waste of cellular metabolism, has now received increasing attention because it was found to directly participate in the pathogenesis of many human diseases including neurological disorders. On one hand, low levels of UA are detrimental to the neurons because of its induction it impairs antioxidant capacity in the cell. High levels of UA, on the other hand, lead to an inflammatory response contributing to gout or neuroprotection. In this review, we summarize this biphasic function of uric acid and highlight potential therapeutic targets to treat UA-related neurological diseases. Despite great progress in understanding the molecular mechanisms underlying human neurodegenerative diseases, the prevalence of nervous system disorders increases, including Alzheimer’s disease (AD), Huntington’s disease (HD), Parkinson’s disease (PD), and Multiple Sclerosis (MS). Uric acid (UA) is a particularly interesting molecule that may be involved in the pathogenesis of AD, HD, PD, and MS –4. AD is5H4N4O3 -trione). It has a molecular weight of 168 Daltons. UA is converted from two nucleotides, adenosine monophosphate and guanine monophosphate. Adenosine monophosphate is converted to inosine by two different mechanisms; either initially removing an amino group by deaminase to form Inosine Monophosphate (IMP) followed by dephosphorylation by a nucleotidase to form inosine, or initially removing a phosphate group by a nucleotidase to form adenosine followed by deamination to form inosine. Guanine monophosphate is converted to guanosine by nucleotidase. Then inosine and guanosine are further converted to hypoxanthine and guanine, respectively, by Purine Nucleoside Phosphorylase (PNP). Both hypoxanthine and guanine form xanthine, and then xanthine is oxidized to form UA. In physiologic conditions, UA is excreted in urine.UA is the end product of purine metabolism. The formula of UA is CHigh serum levels of UA, or hyperuricemia, is defined as a metabolic pathology with a blood UA concentration greater than 7.0 mg/dL in men and 6.0 mg/dL in women. Renal disease is one of the causes leading to hyperuricemia. Babies often have hyperuricemia because they are born with nephrons fewer than healthy adults [It is well known that hyperuricemia is the major etiology of gout in adults. Gout is an inflammatory medical condition characterized by painful, red, tender, hot, and swollen joints, which is caused by the deposition of Monosodium Urate  in the joints, tendon, kidney and other tissues. The deposition subsequently activates caspase-1/inflammasome complex and increases the secretion of caspase-1 substrates including proinflammatory cytokines interleukin-1β (IL-1β) and IL-18 , an impoWhile hyperuricemia has been linked with gout, clinical observations disclosed reduced serum UA levels in some neurological diseases –16 Figu includin2−) and peroxynitrite [Several studies have suggested a causal role of UA in the neurodegenerative diseases. The possible neuroprotective role of UA has been shown by its anti-oxidative action of the non-crystal form of UA because; oxidative stress plays a critical role in neurodegeneration including dopaminergic degeneration in PD ,18. As aynitrite . UA can ynitrite . In addiynitrite . UA stimynitrite . Notablyynitrite . Adminisynitrite . Interesynitrite . Thus, i+ and CD11b+ cells as important mediators in MSU-induced inflammation. In addition, more studies have been focused on the relationship between pathogen-associated molecular Pattern Recognition Receptors (PRRs) and MSU. MSU induces the activation of Nucleotide-Binding Oligomerization Domain (NOD)-like receptor protein 3 (NLRP3), a type of PRRs, and leads to assembly of a protein complex termed inflammasome. The NALP3 inflammasome is composed of NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ACS), and caspase-1, whose function is to activate proinflammatory caspase-1. It processes pre-caspase-1 precursor into its p20-p10 heterodimer activated form, which cleaves and activates the well-known proinflammatory cytokines IL-1β and IL-18. Moreover, amyloid β associated with AD was also found to activate NLRP3 inflammasome-caspase-1 pathway [More recent studies highlight the pathogenic property of UA that induces an inflammatory response contributing to gout and possibly UA-related CNS inflammatory diseases. Blocking antibodies against CD16 and CD11b selectively inhibit the activation of neutrophils and monocytes induced by MSU , indicat pathway . A pilot pathway , suggestA few hypothetical mechanisms have been proposed to explain how a solid structure such as MSU can activate, post-translationally, intracellular receptors like NLRP3. One model suggests that the NLRP3 inflammasome is able to sensitize ionic perturbations induced by MSU crystals, especially potassium efflux . It remaSince MSU is a direct and potent inflammasome activator, it is conceivable to speculate that high levels of UA may induce CNS inflammation and contribute to neurological disorders as well. Although hypouricemia has been associated with several neurological diseases, it has also been found that high levels of UA, which activates the NLRP3 inflammasome, are upregulated in the cerebrospinal fluid of MS patients . Thus, hIn summary, hyperuricemia has long been well known with gout. More recently, hyperuricemia has also been postulated to play a role in neurological diseases. Emerging studies of the pathological mechanisms of gout, pseudogout, and its relationship with neurological diseases have provided insight into the underlying mechanisms of MSU in gout, calcium pyrophosphate dehydrate in pseudogout, and amyloid β in AD as induced NLRP3 inflammasome/caspase-1 activation-mediated inflammatory pathologies. MSU crystal is recognized by NLRP3, which triggers the release of active proinflammatory cytokine IL-1β through NLRP3 inflammasome activation. The pro-inflammatory role of hyperuricemia provides the rationale for targeting NLRP3 and IL-1β as a potential disease-modifying therapy for neurological disorders. More questions remain to be addressed and await answers. If clarified and confirmed, UA could be an important predictor and a potentially modifiable factor affecting the rate of progression in neurological degeneration.


This paper provides a review of fertility research in advanced societies, societies in which birth control is the default option. The central aim is to provide a comprehensive review that summarizes how contemporary research has explained ongoing and expected fertility changes across time and space . A secondary aim is to provide an analytical synthesis of the core determinants of fertility, grouping them within the analytical level in which they operate. Determinants are positioned at the individual and/or couple level (micro-level), social relationships and social networks (meso-level); and, by cultural and institutional settings (macro-level). The focus is both on the quantum and on the tempo of fertility, with a particular focus on the postponement of childbearing. The review incorporates both theoretical and empirical contributions, with attention placed on empirically tested research and whether results support or falsify existing theoretical expectations. Attention is also devoted to causality and endogeneity issues. The paper concludes with an outline of the current challenges and opportunities for future research. Fertility has been a central topic of research within the discipline of demography, but has also achieved considerable interest within sociology, anthropology, economics, medicine and psychology. During the last two decades, research about fertility in advanced societies—societies in which birth control is the default option—has flourished. It is not surprising, therefore, that several reviews of the existing fertility literature have been undertaken ? A secondary aim is to provide an analytical synthesis of the core determinants of fertility, grouping them within the analytical level at which they operate. Determinants are positioned at the micro-level, including determinants at the individual and/or couple level; the meso-level, which encompasses social relationships and social networks ; at the macro-level of the cultural and institutional settings where individuals and couples are embedded. This article not only provides a contemporary review of research and delineation of determinants, but extends our knowledge by adopting an analytical strategy to categorize these determinants, with the aim to provide a better understanding of the often highly interdisciplinary and complex task of explaining fertility trends and differences.We likewise extend existing reviews by adopting a broader multidisciplinary approach, which takes into account relevant contributions from different disciplines beyond those that have been examined previously . We also embrace the most contemporary advances in the field, with attention to the recent fertility reversal in advanced societies. We acknowledge that the topic is highly interdisciplinary, with the term ‘fertility’ often taking on different meanings across disciplines. Subject areas are also diverse with research that examines non-human and non-animal fertility, mainly within the biological and environmental sciences. We focus only on human fertility within advanced societies, and cover research primarily within the disciplines of demography, sociology, medicine, biochemistry, genetics, molecular biology, economics and econometrics, psychology, decision and health sciences.tempo  and quantum . When examining tempo, we know that OECD countries have witnessed a rising mean age at first birth since the 1970s, coupled with an increasing proportion of births among mothers at advanced ages, albeit with considerable country-level variation . While demographic transition theory implies that the quantum of fertility would stabilize around the replacement level of around 2.1 children per woman  total number of children. Timing has always had an important influence on quantum, which is largely a measurement issue, since some of the measures that are used to study the quantum of fertility are not exclusively affected by changes in the timing of fertility. The most well-known case, for instance, is the TFR. Even at the micro-level there is still an influence of timing on quantum, since ‘postponement’ or earlier (perhaps unplanned) births are likely to affect the total number of children in some way. This is particularly relevant when studying the intended parity-progression over a certain time interval (a growing topic during the recent years). For these reasons, we will also highlight the interaction between tempo and quantum.The remainder of this article is structured as follows. Section Research at the micro-level focuses on the individual and/or couple decision-making process underlying the choice to have a child and investigates which circumstances affect decisions regarding the timing and number of children. Choices are often characterized as a rational response to uncertainty and/or as conformity to the prescribed ‘sequencing’ of life course events. A rich body of research has studied the link between life course circumstances and trajectories  and fertility behavior. Other research has instead focused on the role of social class and family or origin, which in turn shapes an individual’s values and preferences. A major obstacle in this line of research is the challenge to establish causality, or in other words, the ability to empirically determine whether these life course factors are actual determinants of fertility or whether certain life course factors and fertility behavior are simultaneously affected by the presence of other common determinants .fertility intentions as a proximate determinant for actual fertility behavior, examining which factors influence the formation, realization and/or change in fertility intentions ; and, (2) parity-progression intentions  child at all or within a specific time frame). Quantum intentions have been shown to be a rather poor predictor of the total actual or realized number of children  Changes in partnership dynamics experienced in the past decades across advanced societies has been linked to the postponement of parenthood. A growing number of studies has shown a parallel tendency to delay union formation and parenthood stability and/or low relationship quality on childbearing. Although causal links are complex and there are important feedback mechanisms  has attracted considerable research interest in economics. Depending on the economic model that is adopted, the effect might be different. The family economics approach, pioneered by Becker , maintaind Lewis  and Willnd Lewis   time, fertility is more costly for higher-income mothers, who are therefore expected to have fewer children  uncertainty are reminiscent of Easterlin’s  theory oKreyenfeld  finds thpreferences, which several authors emphasize, are shaped early in an individual’s life. Catherine Hakim’s Preference Theory  with approaches from behavioral and molecular genetics, neuro-endocrinology, and evolutionary theory  and age at first birth , reversed, or the result of an individual’s simultaneous choice in the two life spheres. Therefore, in order to properly estimate the effect of education  on the age at first birth, potential endogeneity issues must be taken into account , the authors find a non-linear effect of children on happiness, especially for women. Women’s happiness increases after the first child, but having higher-order children is not associated with further increases in well-being. In Europe, parents have been found to be generally happier than non-parents  fertility has focused on ex-communist Eastern European countries. This region has undergone a period of extreme socio-economic uncertainty after the end of the Soviet Union. Bühler and Philipov  provide Bühler and Fratzcak  find a pThe U.S. literature focuses on the support that kin provide to mothers, including childcare and help with raising children, especially for single mothers—here kin are seen as providing ‘safety nets’. This is particularly significant given the high rates of partnership instability and single parenthood Swartz  and is aThe acknowledgement of the importance of social interaction in explaining observed fertility patterns has not yet, however, been coupled with a convincing body of empirical research. The central reason rests with the lack of suitable data and the difficulty to model and properly identify social interaction effects Manski , 1995. TFinally, it is essential to note that the association between the place of residence and fertility choices raises causality concerns, since the relation might be spurious and the effect can work in both directions.A vast literature focuses on how the cultural and institutional setting in which individuals and couples are embedded affects their fertility decision-making. Here an ‘economy versus culture’ dichotomy can be identified Billari . WhereasPeriod effects of economic trends on fertility are usually investigated by linking the TFR to macroeconomic indicators . As mentioned in the introduction, despite the fact that the influence of the timing of fertility on the TFR is mainly a measure of fertility quantum, it does incorporate timing aspects as well. The relationship between TFR and GDP is ambiguous, as Sobotka et al.  demonstrOther studies, however, find contrasting results. Butz and Ward  suggest A related approach that has recently received great attention is the one that explains fertility patterns and cross-country fertility differences in terms of socio-economic development. Myrskylä et al.  provide Instead of looking at GDP, other researchers maintain that indicators of consumer confidence are a better measure of economic recessions, because they reflect the subjective perception of crisis. Van Giersbergen and de Beer  and Fokkunemployment trends on the TFR. Findings consistently showed a negative association: the higher the unemployment, the lower the quantum of fertility  and TFR, showing that in OECD countries, this association has changed from negative (where countries with higher LFP had lower TFR) to positive during the 1980s. Benjamin , Pampel Another approach on the effects of economic trends has been developed by Easterlin , 1968. AA second stream of research has studied the impact of policy measures  on the timing of parenthood, as well as on fertility quantum. There is mixed evidence regarding the effectiveness of social policies on fertility Neyer . GauthieA large number of studies investigate the effects of childcare provision on fertility. Most of the empirical research shows mixed findings. While some studies find that regions with poor childcare coverage have higher fertility , such as Nordic socio-democratic countries, or market provision , enable higher fertility. Conversely, familistic states , where the majority of the economic and caring responsibilities rest on the family, where institutions also support a traditional division of the domestic labor , developed by Lesthaeghe and van de Kaa  to interAs highlighted by van de Kaa , such idage deadlines for childbearing  , promising new and innovative research has focused on how the gendered division of labor, family composition , preferences and intergenerational transmission of values and behavior impact fertility. Although there have been recent advances in including new topics such as the biological and genetic underpinnings of fertility and new family forms, considerable challenges for future research still remain. The first is the availability and affordability of data with sufficient information such as biomarkers or genetic data, but also data that properly captures new types of family forms. Although growing, this type of data that combines genetic and social survey data remains limited. A second related issue, particularly for the introduction of serious biodemographic research, is the need to collaborate with experts and properly understand how to properly integrate this type of information and biological mechanisms in our theoretical, but also statistical models.This review also highlighted core meso-level factors impacting fertility, including the emerging field of social interaction, social capital and networks and place of residence. Although a growing number of  studies started to address these meso-level factors, core challenges still remain. As noted previously, there is no large-scale quantitative network data that has been collected to examine how social networks impact fertility. Of the data that has been collected, the network measurements remained limited. This is partially attributed the high costs of collecting such data, but also the high respondent burden when gathering this type of information, which makes it difficult to include within an existing survey.At the macro-level we summarized the key determinants that have been studied, ranging from economic and (un)employment trends, to policy measures, welfare regimes, value and attitude changes, historical and cultural continuities, contraceptive use and new reproductive technologies. As we noted previously, a key challenge for the credible integration of these macro-level factors for understanding fertility is the need to move from purely theoretical discussions to more convincing empirical tests of this link. Although researchers often claim that macro-level factors such as the welfare regime constellation or societal values impact fertility (and other demographic) behavior, there are few successful empirical attempts to empirically underpin these claims. At all levels we also addressed challenges related to reverse causality and confounding factors and for macro-level factors, the issue of the endogeneity of social policies and reverse causality, which will be discussed in more detail shortly.Some more general problems of current research that we can draw from a broader reading of this review can provide opportunities in helping us to understand improvements for future research. Two problems that became apparent during this review were the clear boundaries between disciplines and geographical areas. First, research on fertility is highly multidisciplinary, i.e., researchers from several disciplines engage in explaining the timing and quantum issues surrounding fertility. However, there are limited instances of interdisciplinary research, simultaneously involving scholars from different disciplines or adopting theoretical and/or methodological approaches of different disciplines. Citation patterns are highly disciplinary-specific, with articles often ignoring clearly relevant research published from other disciplines. It is easy to say that research on fertility would highly benefit by crossing disciplinary boundaries more often, perhaps starting from reading each other’s research more often.A second related point is the relevance of geographic boundaries. Research on fertility on Europe (mostly conducted by European scholars) and research on fertility on North-America (mostly conducted by North-American scholars), or in other words, the bulk of research on fertility, often do not communicate with one another. This was apparent during several places during our review where conflicting theories and findings were presented from North-American and European scholars. Topics, approaches (including the type of data) and again citations remain somehow separated, albeit research in Europe has clearly been fundamental in illuminating the role of macro-level factors, largely due to the often cross-national comparative approach. Not surprisingly, scholars working on other advanced areas are more successful in bridging the two continents over the Atlantic. Also, here it is easy to say that a general understanding of fertility choices would be easier by bridging the findings and approaches related to all advanced societies.Two additional problems are related to methods, data and analytical strategy, which once again are apparent when we stand back to reflect in more general terms from this literature review. First, and related to the international character of fertility research, despite efforts of developing comparative, mostly aggregate data (such as the Human Fertility Database by the Max Planck Institute for Demographic Research and the Vienna Institute of Demography), most research focuses on micro-data that do not usually permit highly comparable research. We could therefore improve fertility research by developing comparable data collection in many countries—including very importantly, the U.S. and Japan in micro-level comparative fertility research. This is even more important given the increasing geographical mobility across countries. Only further collaborative efforts by researchers and funding agencies will enable us to uncover fundamental mechanisms operating at different levels and affecting fertility choices. Second, given that fertility can only be observed  by researchers, the issue of causation versus spurious association lingers as a major problem. Attention to causality is heterogeneous in the literature—undoubtedly with an advantage for studies arising from the tradition of economics. Causal interpretations are widespread also in studies that discover associations (which is a serious problem), but some studies clearly do not aim at understanding or studying causation (which is a lower-order problem related to how ambitious researchers and disciplines are). Further steps towards recognizing the importance of methods aimed at unraveling causality in observational data would contribute to conducting higher-quality fertility research. Researchers and policy-makers alike would gain much more by adopting a program evaluation perspective for the evaluations of policies that might affect fertility choices.Furthermore, our review uncovered that three problems emerge concerning the actual factors studied, ranging from individuals to context. Research on men, or in other words, the fertility of men and fatherhood, remains very limited, albeit growing. It is clear that a gendered approach is necessary, but this implies that both genders should play an equal role in our understanding of fertility choices. More research including both men and women would improve our knowledge. Related to this first problem is the second problem of couples. For both theoretical and (lack of) data-related reasons, fertility choices have been investigated mostly from an individual perspective. The limited research, and data, existing on couples show the incredible value of addressing fertility as a joint decision. A third problem relates to the limited knowledge of the importance of meso-level factors. Here the theory is more developed than the actual instruments such as the collection of quantitative data having a kinship and/or network-based approach, which we addressed previously. Efforts in using innovative analytical techniques such as agent-based modeling are promising. Recent innovative designs  also provide some insights on future directions.Finally, promising research avenues are those emphasizing the interaction of factors located at different analytical levels. As already mentioned in the policies and welfare regime sections, studies that adopt a cross-country comparative life course approach often position nation-specific institutions as path-dependent structures that shape micro-level individuals’ characteristics and enable or inhibit the ability to have children and to have them at a particular period in their lives . National institutions or forces such as employment and education systems, welfare regimes, social policies, family, and gender systems are historically based and country-specific and determine the degree to which people are affected by macro-level changes Mayer . Micro-lThroughout this review, we have already cited some studies that adopt a cross-country approach, showing how forces and constraints at the macro-level can impact micro-level dynamics. Kalwij  and BegaMcDonald’s gender theory , 2000b aAnother example of meso-macro interaction is the study of Balbo and Mills . They sh


Human T-lymphotropic virus types I and II (HTLV-I and -II) cause myelopathy; HTLV-I, but not HTLV-II, causes adult T-cell leukemia. Whether HTLV-II is associated with other diseases is unknown. Using survival analysis, we studied medical history data from a prospective cohort of HTLV-I– and HTLV-II–infected and –uninfected blood donors, all HIV seronegative. A total of 152 HTLV-I, 387 HTLV-II, and 799 uninfected donors were enrolled and followed for a median of 4.4, 4.3, and 4.4 years, respectively. HTLV-II participants had significantly increased incidences of acute bronchitis (incidence ratio [IR] = 1.68), bladder or kidney infection (IR = 1.55), arthritis (IR = 2.66), and asthma (IR = 3.28), and a borderline increase in pneumonia . HTLV-I participants had significantly increased incidences of bladder or kidney infection (IR = 1.82), and arthritis (IR = 2.84). We conclude that HTLV-II infection may inhibit immunologic responses to respiratory infections and that both HTLV-I and -II may induce inflammatory or autoimmune reactions. Human T-lymphotropic virus types I and II (HTLV-I and -II) are presumed to have derived from primate T-lymphotropic viruses with which they share significant nucleotide sequence homology Other possible health outcomes of chronic HTLV-I and -II infection have not yet been adequately investigated. Patients with adult T-cell leukemia may develop opportunistic infections such as Pneumocystis carinii pneumonia Case series and cross-sectional studies of HTLV-I and -II disease outcomes are vulnerable to potential bias and confounding. We have prospectively followed a large cohort of former blood donors with well-documented HTLV-I and -II infection at enrollment, and a similar group of uninfected donors, all of whom are HIV seronegative. We report on the occurrence of various disease outcomes in this cohort after a median follow-up of 4.3 years.This study is a prospective, multicenter cohort of persons with HTLV-I and -II infections, which were detected at the time of attempted blood donation at five U.S. blood centers and comparable HTLV–seronegative donors. Details of the cohort enrollment and follow-up procedures have been published previously , or both, as previously described Each visit with a study nurse consisted of an interviewer-administered health history questionnaire and phlebotomy of blood for complete blood count and other studies. Selected diagnoses  reported on the questionnaire triggered requests for confirmatory medical records. We included nine conditions or diseases  and eight symptoms  in the data analysis.We used the Kaplan-Meier product limit method to calculate the unadjusted probability of disease-free survival during the study period for each disease outcome by HTLV status. Survival time was defined as the number of days from the baseline visit until the date that an adverse health outcome was first diagnosed or the end of observation. We performed the log-rank test to assess the differences in disease‑free survival time (days) between HTLV-seronegative participants and HTLV‑I– or HTLV-II– infected participants, respectively.To adjust for possible confounding factors, we performed multivariable analysis with HTLV status as an independent variable, survival time as a dependent variable, and possible confounding factors as covariates. In constructing the survival analysis models, we considered a number of covariates, which are described as follows: demographic variables , education, smoking history , alcohol consumption, blood center, community versus autologous donation type, injection drug use , parity , and number of sex partners (in model for bladder and kidney infections only). Using a backward selection process, all these covariates were added to the initial model, but only covariates with significant independent associations themselves (p < 0.05), or which substantively modified HTLV incidence ratio, were retained in the final model. We did not include interaction terms because similar analyses of data from prior cohort visits had indicated the absence of significant interaction.To examine the differences in the cumulative number of episodes of pneumonia, bronchitis, and bladder or kidney infection by HTLV status, we used the negative binomial model, a generalization of the Poisson model, to compute incidence rate ratios (RR) with 95% confidence interval (CI) for each of these outcomes. This model took into account that the recurrence of the disease in a participant may be associated with both the overall disease incidence and its previous occurrence in that participant. We also adjusted for possible confounding factors using the same modeling strategy as for the survival analyses.For the analysis of symptoms, we calculated unadjusted and adjusted odds ratio (OR) with 95% CI for any occurrence of each symptom by HTLV status by using logistic regression models. To adjust for possible confounding, we added other possible confounding factors as covariates to the models by using the same approach as in the survival analyses. All analyses were performed using Statistical Analysis System (SAS) software During the initial visits in 1990 through 1992, we enrolled 154 HTLV-I, 387 HTLV-II, and 799 HTLV seronegative persons. Two HTLV-I participants were excluded from this analysis because they did not complete the screening physical examination at the initial visit. The baseline characteristics of the study population are given in We present data through the third study visit in 1995 through 1996. Median follow-up time was 4.3 years for all 1,338 participants, including those with no follow-up. Median follow-up did not differ by HTLV status and was 4.4 years for the HTLV-I group, 4.3 years for the HTLV-II group, and 4.4 years for the HTLV-seronegative group.Compared to seronegative persons, HTLV-I–infected persons were more likely to have a new diagnosis of bladder or kidney infection (p = 0.009) and arthritis (p = 0.0002) . Among tThe number of incident cases diagnosed (limited to one case per person) and the unadjusted and adjusted incidence ratios (IR) for several diagnoses are given in To further investigate the occurrence of infectious diseases, we also analyzed the incidence density of three infectious diseases by HTLV status, whether or not each diagnosis was a first or recurrent case . In the Disease-free survival curves for selected medical diagnoses, by HTLV status, are represented in the Figure. Compared to seronegative persons, HTLV-II–infected participants were more likely to experience acute bronchitis (p < 0.0001), bladder or kidney infection (p = 0.0008), arthritis (p = 0.0003), and asthma (p = 0.0007); the likelihood of acquiring pneumonia was increased but not significantly (p = 0.08). Differences between the HTLV-II and HTLV-seronegative participants in the incidence of cancer, hypertension, diabetes, or thyroid disease were not statistically significant.The number of incident cases diagnosed (limited to one case per participant), and the unadjusted and adjusted IRs, for several diagnoses are given in In the incidence density analysis of recurrent infections, an average of 0.21 cases of pneumonia, 1.10 cases of acute bronchitis, and 1.25 cases of bladder or kidney infection occurred among HTLV-II participants during their 4.3-year median follow-up time . The corIn summary, HTLV-II–infected participants had a higher incidence of acute bronchitis, bladder or kidney infection, arthritis and asthma, and a higher incidence of pneumonia than did HTLV-seronegative participants followed concurrently. The finding of a higher rate of these infectious diseases among HTLV-II participants was supported both by survival analysis, which considered only the first diagnosis, and by negative binomial modeling, which considered both first and recurrent infections. HTLV-I participants had a higher incidence of bladder or kidney infection and arthritis. Cancer incidence was not higher in either the HTLV-I or HTLV-II participants compared to its incidence in seronegative participants, although the number of cases was small.Our finding of a higher incidence of respiratory tract infections among HTLV-II–infected persons is consistent with most of the small number of other published clinical studies on this topic. Robert-Guroff et al. found higher rates of HTLV-II infection among injection drug users with abscess than in those without abscess The biologic basis for a putative increased susceptibility to certain infections in humans with chronic HTLV-II infection is not well described. In contrast to the predominant CD4+ lymphotropism of HTLV-I, HTLV-II provirus in vivo is integrated at highest levels into CD8+ lymphocytes but may also be demonstrated in CD4+, both CD45RO+ and CD45RO-, and even non-T  lymphocytes Our finding of an association of both HTLV-II and HTLV-I with bladder or kidney infection is consistent with a previous report of unspecified renal disease in a prospective cohort study of HTLV-I–infected persons in Japan The increased incidence of arthritis observed for both persons infected with HTLV-I and HTLV-II supports reports of possible autoimmune syndromes with HTLV infection. HTLV-I has been epidemiologically associated with uveitis We have previously reported a single case of adult T-cell leukemia which was diagnosed between the first and second visits of the patient in this cohort study The two- to three-fold higher prevalence of self-reported neurologic symptoms, including trouble walking, climbing stairs, or rising from a chair, and bladder symptoms may represent early spinal cord injury due to HTLV-I or -II. As follow-up of the cohort continues, we shall be able to determine whether those reporting such symptoms in earlier visits have a higher incidence of overt myelopathy than asymptomatic HTLV-infected participants. On the other hand, the frequency of self-reported lymphadenopathy and night sweats is unlikely to be caused by preclinical hematologic malignancy, given the rarity of that disorder in HTLV-I–infected persons and its lack of association with HTLV-II. These symptoms might be related to known effects of HTLV-I and -II on lymphocytic proliferation and cytokine expression, or they might simply reflect reporting bias.Strengths of the current study include its controlled, prospective, cohort design, stringent confirmation of HTLV-I– and -II–infection status at baseline, and systematic ascertainment of disease outcomes. One potential weakness is that differences in socioeconomic status and risk behaviors could have confounded disease associations between HTLV–infected and uninfected previous blood donors, even though we selected the uninfected participants in strata defined by the age, sex, race or ethnicity, center, and blood donation type of the HTLV groups. We controlled for the socioeconomic and behavioral factors using multivariate analyses, but residual confounding could affect the magnitude of the associations we observed. Second, recall bias may exist in that participants with HTLV infection might differentially report more diagnoses because of heightened concern about their own health. Our questionnaire requested only medically confirmed diagnoses, and the absence of associations with noninfectious disease, such as hypertension, diabetes, and thyroid disease, suggests that generalized overreporting of illness was not a problem. Additionally, infectious disease associations with HTLV have not been widely reported, so we do not think that recall bias specific to these diagnoses was a serious concern. Finally, follow-up time to date is modest for a chronic infection such as HTLV, and our findings may change with longer observation.In conclusion, HTLV-II infection is associated with an increased incidence of respiratory and urinary tract infections and asthma, and both HTLV-I and -II are associated with increased incidence rates of arthritis, compared with results for seronegative persons. These findings suggest that chronic infection with HTLV-II may inhibit host immunologic responses to infection, or more specifically, to respiratory infections. The arthritis and asthma results, and possibly the respiratory tract diagnoses, suggest that other inflammatory or autoimmune reactions may be induced by HTLV-I or HTLV-II infection. Additional in vitro and in vivo research on the immunologic consequences of HTLV infection is needed.