There is significant evidence from murine and human genomic studies that B-cell receptor (BCR) signaling abnormalities are potential factors in the pathogenesis of lupus. However, data on signaling deviations in lupus patients are scant. We have undertaken a project to comprehensively study BCR signaling deviations in lupus patients.+IgG+), mature naïve , transitional , B1  and IgM memory B cells . We will measure pERK levels at baseline (time 0), 1 minute , 5 minutes  and 15 minutes . We will establish normal parameters by studying BCR signaling in 100 nonautoimmune individuals. We will then determine the prevalence of low and high BCR signaling deviations in 100 lupus patients. Signaling deviations will be correlated with clinical data.Peripheral blood mononuclear cells will be isolated and frozen. B cells will be stimulated with F(ab')2 anti-IgM and anti-IgG. Using eight-parameter flow cytometry, we will determine signaling amplitude, as measured by phosphorylated ERK1/2, in IgG memory cells (CD20We have optimized a protocol that enables us to identify each of the above B-cell subsets while assaying phospho-ERK levels. Results from four lupus patients indicate that this protocol is able to identify signaling differences between individuals with lupus. See Figure The extent to which signaling deviations contribute to autoimmunity in patients with lupus remains to be determined. By elaborating a robust method to assay BCR signaling, we hope not only to measure this pathogenic factor but also to answer fundamental questions, such as whether diminished BCR signaling correlates with a broader autoimmune phenotype, as would be postulated by the impaired negative selection that is caused by diminished signaling. In the future, we will aim to convert signaling studies from research tools to clinical tools.


Most people infected with Human T-cell Lymphotropic Virus Type 1 (HTLV-1) remain clinically asymptomatic; however, a minority develops the debilitating myelopathy HAM/TSP. Current treatment of HAM/TSP is limited by our partial understanding of the protective immune response to HTLV-1 and the pathogenesis of HAM/TSP.We wished to test the hypothesis that a gene expression signature in peripheral blood distinguishes between patients with HAM/TSP and ACs. We investigated genome-wide transcription patterns in whole blood from HTLV-1 asymptomatic carriers , patients with HAM/TSP (n=20) and uninfected control subjects (n=17). We identified a 542-gene signature that was deregulated in all HTLV-1+ individuals and predominantly comprised transcripts involved in p53-mediated DNA damage responses (p=0.00489). An 80-gene signature distinguished patients with HAM/TSP from those with the clinically similar disease multiple sclerosis. Paradoxically, at a given proviral load patients with HAM/TSP, but not ACs, over-expressed antiviral interferon-stimulated genes .Expression of these ISGs (assessed by quantitative PCR and flow cytometry) was not limited to HTLV-1-infected CD4+ T cells, suggesting that all peripheral blood immune cells were exposed to interferons (IFN) in vivo. Neither elevated IFN plasma levels nor an abnormal capacity for IFN production was detected in patients with HAM/TSP. However, peripheral immune cells in patients with HAM/TSP were more sensitive to IFN-alpha and IFN-gamma stimulation.These findings suggest that chronic over-expression of a specific subset of ISGs is ineffective in containing HTLV-1 and may instead contribute to the pathogenesis of HTLV-1-associated myelopathy.


Studies on human fertility genes have identified numerous risk/protective alleles involved in the occurrence of reproductive system diseases causing infertility or subfertility. Investigations we carried out in populations at natural fertility seem to suggest that the clinical relevance that some fertility genes are now acquiring depends on their interaction with contemporary reproductive behaviors . In recent years, a new physiological role in human fertility regulation has emerged for the tumor- suppressor p53 gene (P53), and the P53 Arg72Pro polymorphism has been associated with recurrent implantation failure in humans. To lend support to our previous observations, we examined the impact of Arg72Pro polymorphism on fertility in two samples of Italian women not selected for impaired fertility but collected from populations with different (premodern and modern) reproductive behaviors. Among the women at near-natural fertility (n = 98), the P53 genotypes were not associated with different reproductive efficiency, whereas among those with modern reproductive behaviors (n = 68), the P53 genotypes were associated with different mean numbers of children  and a significant negative relationship between the number of children and P53 Pro allele frequencies (p = 0.028) was observed. These results are consistent with those of clinical studies reporting an association between the P53 Pro allele and recurrent implantation failure. By combining these findings with previous ones, we suggest here that some common variants of fertility genes may have become “detrimental” following exposure to modern reproductive patterns and might therefore be associated with reduced reproductive success. Set within an evolutionary framework, this change could lead to the selection of a set of gene variants fitter to current reproductive behaviors as the shift to later child-bearing age in developed countries. Human fertility is a complex trait determined by gene-environmental interactions in which genetic factors represent a significant component in vitro fertilization (IVF) or other assisted reproductive technology (ART) procedures Different alleles at a locus are usually thought to be associated only with small fertility differences  and contribute very little to overall fitness, otherwise selection would lead to their fixation or elimination http://ec.europa.eu/employment_social/social_situation/docs/sec_2007_638_en.pdf; last accessed February 23, 2012).Whether for economic, social, or educational reasons, young couples have begun to postpone starting a family and reduce family size, and women to delay having first childbirth or decide to remain childless. In Western Europe, the mean age at which women deliver their first child rose from 22–25 years in 1950 to 27–30 years in 2003 To verify the hypothesis that exposure to different reproductive behaviors may influence the action of fertility-related genes, we examined the role of another fertility gene, P53, in two samples of the Italian population with different reproductive patterns. The tumor-suppressor p53 gene (P53) has a pivotal role in tumor prevention and DNA damage response. In response to a variety of cellular stresses, the p53 protein (p53) is able to activate target genes to induce cell cycle arrest, apoptosis, and senescence. Through these mechanisms, p53 promotes DNA repair or the elimination of damaged cells, thus contributing to tumor suppression In the last years, a new physiological role has emerged for p53 as a fertility regulator in vitro studies: the P53 Pro72 allele was found at increased frequencies among women undergoing in vitro fertilization (IVF) and, among such women, it was associated with higher implantation failure Many single nucleotide polymorphisms (SNPs) of P53 have been identified in human populations, but the one most extensively studied is rs1042522, located in codon 72 of exon 4, where the change CGC→CCC results in an Arg/Pro substitution. The Pro and Arg alleles at codon 72 are not functionally equivalent: Pro72 encodes a weaker p53 isoform than that encoded by Arg72 to induce apoptosis and suppress transformation, while it is better at favoring cell cycle arrest So far, the P53 Arg72Pro polymorphism has been found to influence human reproduction in a pathological context. We examined it in two samples of women not selected for impaired fertility and collected from populations with different reproductive patterns. The first was collected from a rural population from Southern Italy, which, according to population and sample fertility rate, has reproduced with a pre-modern or near-natural reproductive pattern. The second was collected from an urban population from northeastern Italy whose fertility rate indicates a pattern of reproduction typical of modern societies where birth control is practiced.The aim of the present study was to examine the role of P53 polymorphism in fertility at the population  level and the possible effects different reproductive behaviors may have on it.http://www3.istat.it/dati/catalogo/asi2004/PDF/Cap3.pdf, last accessed February/27/2012].The characteristics of the samples are reported in The P53 Arg72Pro polymorphism was examined in the Southern Italian (n = 152) and the Northern Italian (n = 102) samples. The observed genotype frequencies  agreed with those expected according to the Hardy-Weinberg equilibrium. The P53 Pro allele frequencies of the two samples were 0.250  and 0.303 , similar to those reported for the Italian population A statistically significant result was achieved with a different approach: the subjects were grouped into different classes according to the number of children, and the P53 allele frequencies were calculated for each class . The chiThis study examined the association of the P53 Arg72Pro polymorphism with fertility at the population level, specifically in women not selected for fertility disorders. The data show that in the sample of women at near-natural fertility, the P53 genotypes are not associated with differential fertility. Conversely, in the sample of women with modern reproductive behaviors, the P53 genotypes are associated with a different reproductive efficiency, with the lowest fertility occurring among the Pro/Pro genotype carriers. The observation of a reduced fertility associated with the Pro allele was confirmed by subsequent chi square for trend of proportions test that shows a significant inverse relationship between Pro allele frequency and classes according to number of children.These results should be interpreted with caution. The size of the samples is relatively small and, fertility being a complex trait, numerous other genetic and environmental factors, including male fertility, could have contributed to determine the phenotype “number of children” we examined. Nevertheless, the results obtained in the sample of women with a modern reproductive pattern are consistent with those of clinical studies reporting an excess of P53 Pro/Pro genotypes in women with unexplained infertility undergoing IVF and, among these, in women with higher implantation failure It is interesting to observe how the phenotypic effects of P53 genotypes vary according to reproductive environments. In women from the population at near-natural fertility, P53 genotypes do not influence fertility, suggesting that the possible small reduction of fertility associated with the P53 Pro allele could have been offset by the pre-modern reproductive behavior . In the sample from the population where modern reproductive behaviors began to be followed, the P53 genotype effect begins to shift toward differential fertility. Set within the broader context of contemporary society, the role of P53 genotypes eventually tends to acquire clinical relevance, being associated with infertility and the successful/or not outcome in women undergoing IVF pp and xx) influenced the fertility level, predisposing to have an elevated number of children . In the Italian women, the same genotypes showed an association with a low rate of spontaneous abortions, but without any impact on total fertility. In today's industrialized societies, however, the effects of the ESR1 genotype manifest as a successful outcome in women undergoing IVF These findings are similar to what we have observed for other fertility-related genes. One gene we studied was ESR1, which encodes estrogen receptor alpha Another example is the angiotensin I-converting enzyme (ACE) gene. Recently, the ACE I/D polymorphism was found associated with recurrent spontaneous miscarriages The emerging picture of the interactions between fertility genes and contemporary reproductive behaviors suggests that fertility genes may represent a further example of genes “rendered detrimental by progress”. The hypothesis for a “genotype rendered detrimental by progress” was first advanced by J.V. Neel Taken together, our data seem to indicate that fertility genes might easily fit within the “altered lifestyle syndromes” hypothesis, and, at least for the P53 and ACE genes, in the ancestral susceptibility model as well, given that both the Pro allele of P53 and the D allele of ACE are the ancestral alleles On the whole, the picture we have outlined suggests the occurrence of complex interactions between genetic factors and reproductive environments and that fertility genes are a further set of genes whose functions are influenced by the lifestyle changes of modern societies.The Southern Italian population sample included 152 apparently healthy unrelated women in the post-reproductive age collected for a population survey of the elderly population of Cilento, a rural area in the district of Salerno, Southern Italy. All subjects were born before 1930 (1900–1930) and lived in a society where the demographic transition (increase in life expectancy and declining fertility) had just started The Northern Italian population sample comprised 102 healthy unrelated women recruited as a control sample during an epidemiological study on Alzheimer's disease (AD) carried out at the Verona Hospital. All subjects were born between 1921/1953 in the urban area of Verona, northeastern Italy, and lived in a society where the demographic transition was already under way. In the Italian women born in the same geographic area in the same year range, the mean age at marriage was 25.2 years and the fertility rate was near/below the replacement level  58-GTCCCCCTTGCCGTCCCA-38, and Arg–, 58-CTGGTGCAGGGGCCACGC-38, amplifying a 137-bp fragment; P53 Pro sequences were detected using the primer pair Pro1, 58-GCCAGAGGCTGCTCCCCC-38 and p53–,8-GGAAGCCAGCCCCTCAGG-38, amplifying a 206-bp fragment. Visualization of the PCR product on agarose gel revealed three different genotypic patterns.Venous blood was drawn in ethylenediaminetetraacetic acid (EDTA) from all subjects after overnight fasting. High-molecular-weight DNA was extracted from whole blood according to the procedure described by Miller et al. Allelic frequencies were determined by the gene-counting method. The agreement of the genotype distribution with Hardy–Weinberg expectations was verified with the chi square test. Comparisons between the mean number of children associated with different P53 genotypes were performed using analysis of variance (ANOVA). Estimates of statistical power (1-β) were calculated according to Cohen


Much of the mechanistic details for how HTLV-1 Gag orchestrates virus particle assembly and release are poorly understood. Here, we monitored the behavior of both membrane-bound and cytoplasmic HTLV-1 Gag in real-time in living cells incubated on a fluorescence microscope. We used both fluorescence fluctuation spectroscopy  and fluorescence imaging ) to investigate the relationship between cytoplasmic and membrane bound Gag, using a Gag-YFP model system. FFS determines the brightness, mobility, and concentration  and localization (z-scan) of fluorescent particles from the intensity bursts generated by individual particles passing through a small observation volume, which yields information about protein stoichiometry, interactions, transport, and distribution. By coupling the single-molecule FFS technique with imaging techniques capable of monitoring Gag localization (epi-illumination) and membrane-specific localization (TIRF), we achieved new insights into the earliest events in HTLV-1 Gag assembly, and differences to HIV-1 Gag. We found that HTLV-1 Gag membrane-targeting occurred at all cytoplasmic concentrations measured, while appreciable membrane-targeting for HIV-1 required Gag cytoplasmic concentration to exceed a threshold. In addition, z-scan FFS revealed that a substantial population of membrane-bound HTLV-1 Gag exists not as puncta, but as a diffuse, low-order, dynamic “sheet.” These observations, coupled with previous observations of cytoplasmic Gag interactions and mobility, point to differences in membrane targeting of HTLV-1 and HIV-1 Gag. In summary, the suite of biophysical fluorescence techniques, applied HTLV-1 Gag, provide unparalleled information concerning HTLV-1 Gag trafficking processes in vivo, elucidating assembly pathway differences between HTLV-1 and other retroviruses.


Entamoeba histolytica is an intestinal protozoan parasite that causes amoebiasis in humans, affecting up to 50 million people worldwide each year and causing 40,000 to 100,000 deaths annually. EhCP112 is a cysteine proteinase of E. histolytica able to disrupt cell monolayers and digest extracellular matrix proteins, it is secreted by trophozoites and it can be active in a wide range of temperature and pH. These characteristics have encouraged the use of EhCP112 in the design and production of possible vaccines against amoebiasis, obtaining promising results. Nevertheless, we have no conclusive information about the role of EhCP112 in the E. histolytica pathogenesis.Ehcp112 gene via the soaking system. Silencing was evaluated by Western blot using an antibody against the EhCP112 recombinant protein. Finally, we analyzed the protease activity, the phagocytosis rate and the ability to destroy MDCK cells of the EhCP112-silenced trophozoites.A set of three specific siRNA sequences were used to silence the in vitro virulence assays, which showed that EhCP112 silencing produces a significant reduction in cytolysis and phagocytosis of target cells, indicating the participation of this proteinase in these events.The highest silencing effect on EhCP112 was detected at 16 h of treatment; time enough to perform the in vitro virulence of E. histolytica.EhCP112 is involved in the  Entamoeba histolytica is the intestinal protozoan parasite that causes amoebic colitis and amoebic liver abscesses in humans [n humans , affectin humans . The cytn humans  and seven humans .E. histolytica[in vitro are thought to play a role in infection of the human host, invasion and destruction of host tissue and completion of the parasite life cycle [CPs have been identified as crucial virulence factors of protozoa parasites because they are associated to cytoadherence, haemolysis, cytotoxicity, degradation of extracellular matrix components, evasion of the immune response and nutrient acquisition -10. The stolytica. The genstolytica,23. Thosfe cycle .E. histolytica formed by the EhCP112 cysteine proteinase and the EhADH112 adhesin [E. histolytica has not been proved directly.EhCPADH is a protein complex of  adhesin . This co adhesin . EhCP112 adhesin . The rec adhesin ,27. The E. histolytica trophozoites leads to a reduction of the in vitro virulence of the parasite.Here, we found that RNAi-mediated downregulation of EhCP112 in Trophozoites of clone A , strain Ehcp112 gene fragment (1278 bp), which encodes a polypeptide containing the pro-peptide and the mature enzyme, cloned in the expression vector pTrcHIs C [E. coli, strain BL21 (pLys E). These bacteria were incubated with 1 mM of Isopropyl β-D-1-thiogalactopyranoside (IPTG) for 3 h at 37°C [2+-affinity columns  following the manufacturer’s protocol.An TrcHIs C  was used at 37°C  to inducTo obtain antibodies against rEhCP112, 100 μg of the recombinant protein was subcutaneously inoculated four times at intervals of 15 days on New Zealand rabbits. The first immunization was performed in the presence of complete Freund’s adjuvant (Sigma-Aldrich), and additional immunizations were carried out in the presence of incomplete Freund’s adjuvant (Sigma-Aldrich). The experimental protocol was approved by the institutional committee for animal care and provided all technical specifications for the production, care and use of laboratory animals (NOM-062-ZOO-1999).Ehcp112 mRNA sequence (GenBank: AF127375.2) was analyzed by the online program Target finder [, and by the multiple sequence alignment ClustalW[Entamoeba genome database Pathema [E. histolytica CPs. A set of three specific siRNA oligonucleotide sequences for Ehcp112 were chosen  and then, respective sense and antisense oligonucleotides were hybridized using the 1X DNA Annealing Solution (Ambion) incubating at 90°C for 3 min and then at 37°C for 1 h.6) freshly collected from 90% confluent cultures were inoculated in 25 ml culture plastic flasks (Corning) containing TYI-S-33 medium and incubated at 37°C during 24 h. Then the annealed siRNAs at 25, 50 and 100 μg/ml were added to the cultures and incubated at 37°C for 16, 24, and 36 h. To verify the siRNA entry to trophozoites by this soaking method, 75 pmol of a fluorescein-labeled negative control siRNA (Ambion) were added to 1 × 106 trophozoites during 2, 5 and 16 h and internalization of the labeled-siRNA was analyzed by confocal microscopy. To determine the effect of the EhCP112 silencing on growth, the number of trophozoites and its viability was analyzed by trypan blue staining each 12 h after soaking with siRNA.Trophozoites , followed by incubation with a mouse anti-rabbit IgG secondary antibody conjugated to horseradish peroxidase (Invitrogen) . Finally, the antibody detection was developed by incubation with 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) and H2O2. As internal control, the same membranes were revealed with anti-actin antibodies . The bands detected by the anti-EhCP112 antibodies were analyzed by densitometry and data were normalized with those obtained from bands recognized by the anti-actin antibodies.Total extracts from 7/ml in PBS. The cellular pellets were lysed by five freeze-thaw cycles in liquid N2 and 10 μl of a 1:1000 dilution were mixed with 10 μl of 2× sample buffer without 2-mercaptoethanol and electrophoresed in 10% SDS-PAGE copolymerized with 0.1% gelatin. After electrophoresis, gels were incubated in 2.5% Triton ×-100 for 1 h at room temperature. CPs were activated by incubation in buffer activation  for 12–16 h at 37°C. Gels were stained with 0.25% Coomassie blue R-250 and clear bands were indicative of proteolytic activity.To analyze the protease activity, trophozoites were washed twice with phosphate-buffered saline (PBS) and suspended at concentration of 105 cells) grown in 24-well plates (Costar) were incubated with 1 × 105 trophozoites (cytopathic assays) or with total extracts from 1 × 106 trophozoites (cytotoxic assays) in the presence of 0.02% of 2-mercaptoethanol during 2 h at 37°C. After this time, trophozoites or their extracts were eliminated and the remaining monolayers were washed three times with PBS, fixed with 2.5% glutaraldehyde, and cellular destruction was evaluated as previously described [MDCK (Madin-Darby canine kidney) monolayers (1 × 10escribed . BrieflyE. histolytica[A405 of sample/ A405 of control trophozoites (NRS-treated) at 10 min of phagocytosis. Results were reported as the means ± standard deviation of the percentage of normalized values of three independent experiments by duplicate.Erythrophagocytosis was measured using human red blood cells (hRBCs) as previously described , with sostolytica. Then, tE. histolytica[E. histolytica pathogenesis.EhCPADH is a protein complex formed by a CP (EhCP112) and an adhesin (EhADH112) . This costolytica,26,27,38E. histolytica[E. histolytica pathogenesis, we analyzed the in vitro virulence of EhCP112-silenced trophozoites.The RNAi technology has proved to be a strong tool in molecular biology to study the functional analysis of cellular genes ,40 and astolytica-47. ConsE. coli, strain BL21 (pLys E). In SDS-PAGE of extracts of induced bacteria we observed a slight enrichment of a 52-kDa protein  . Thus, tIn order to generate antibodies against EhCP112, New Zealand rabbits were subcutaneously inoculated with 100 μg of the purified rEhCP112. Then, the antibodies obtained were tested by Western blot. In these assays, two bands were recognized in both the recombinant protein and trophozoite extracts  as target. The online program Target finder  predicte Pathema  and the  Pathema . We elimE. histolytica genes, included other CPs. As a negative control, an additional set of non-related sense and antisense primers were used.A set of three specific EhCP112 siRNA oligonucleotide sequences were chosen  for 16, 24 and 36 h. Each siRNA was tested separately and mixed. In these assays we observed a decreased expression of EhCP112 in trophozoites treated for 16 and 24 h with 50 μg/ml of the siRNA 1, corresponding to the 5′-end of the gen, whereas the EhCP112 expression was not affected by incubation with the siRNA control, containing a non-related sequence (NRS) . In addition, siRNA 2 and siRNA 3 did not show a significant effect in the EhCP112 expression. Consequently, the incubation with the siRNA mixture did not increase the EhCP112 silencing caused by siRNA 1 (data not shown). Then, consecutive experiments were performed with trophozoites after 16 h of treatment with 50 μg/ml of siRNA 1, because in these conditions we observed the most efficient effect on the EhCP112 silencing.in vitro virulence of E. histolytica. First, the protease activity of the silenced trophozoites was analyzed in polyacrylamide-0.1% gelatin substrate gels. The EhCP112-silenced trophozoites showed a 62-kDa band with smaller proteinase activity to that of trophozoites treated with the non-related sequence (NRS) and untreated trophozoites  in relation to the NRS-treated trophozoites Figure B, suggesIn these assays we evaluated the monolayer cell destruction by total trophozoite extracts, where all the amoeba cysteine proteinases have been released. Nevertheless, there are few cysteine proteinases, including EhCP112, reported to be secreted that could be involved in cytolysis ,48. To aE. histolytica[E. histolytica.We also carried out erytrophagocytosis assays, because phagocytosis is an event involved in the pathogenic mechanism of stolytica. Thus, wE. histolytica. However, despite that expression of EhCP112 decreased around 89% after incubation with siRNA, cell cytotoxicity, cytopathic effect and erythrocyte phagocytosis between siRNA and NRS treated trophozoites showed minor, but significant, differences (25 to 34%). These results suggest that other mechanisms might also contribute in these processes.Data obtained in this study support the hypothesis that EhCP112 is involved in the pathogenicity of E. invadens, a related parasite of reptilians, is increased in the cyst stage [Entamoeba sp. The EhCP112 silencing could be useful to test this possibility; unfortunately the in vitro encystation of E. histolytica is not still available.On the other hand, it has been reported that the expression of the homologue of EhCP112 in st stage , suggestin vitro virulence assays, which showed that EhCP112 silencing produces a significant reduction in cytolysis and phagocytosis, indicating the participation of EhCP112 in these events. These results and those obtained with the EhCP112 recombinant protein [E. histolytica, as a therapeutic target or a vaccine candidate.In this study we achieved the silencing of EhCP112 by siRNA. The highest silencing effect was detected at 16 h of treatment; time enough to perform the  protein  support E. histolytica which is part of the EhCPADH protein complex; hRBCs: Human red blood cells; IPTG: Isopropyl β-D-1-thiogalactopyranoside; MDCK: Madin-Darby canine kidney cell line; NRS: Non-related sequence; PBS: Phosphate buffered saline; rEhCP112: EhCP112 recombinant protein; siRNA: Small interference RNA.BLAST: Basic Local Alignment Search Tool; CPs: Cysteine proteinases; EhCP112: Cysteine proteinase of The authors declare that they have no competing interests.ROR and WF conceived and carried out experiments, analyzed data and drafted the manuscript; MBM, ROQ and EO participated in the design of the study, analyzed data and helped to draft the manuscript; MAR conceived and designed the study, analyzed data and drafted the manuscript. All authors read and approved the final manuscript.


Mycobacterium tuberculosis infection and severity of tuberculosis. Although previous studies have shown that HTLV-1 infected individuals have a low frequency of positive tuberculin skin test (TST) and decreasing in lymphoproliferative responses compared to HTLV-1 uninfected persons, these studies were not performed in individuals with history of tuberculosis or evidence of M. tuberculosis infection. Therefore the reasons why HTLV-1 infection increases susceptibility to infection and severity of tuberculosis are not understood.The aim of this study was to evaluate how HTLV-1 may influence the clinical, bacteriologic and immunologic presentation of tuberculosis.HTLV-1 is associated with increased susceptibility to The study prospectively enrolled and followed 13 new cases of tuberculosis associated with HTLV-1 (cases) and 25 patients with tuberculosis without HTLV-1 infection (controls). Clinical findings, bacterial load in the sputum, x-rays, immunological response and death were compared in the two groups.There were no differences in the demographic, clinical and TST response between the two study groups. IFN-γ and TNF-α production was higher in unstimulated cultures of mononuclear cells of case than in control patients (p < 0.01). While there was no difference in IFN-γ production in PPD stimulated cultures, TNF-α levels were lower in cases than in controls (p = 0.01). There was no difference in the bacterial load among the groups but sputum smear microscopy results became negative faster in cases than in controls. Death only occurred in two co-infected patients.While the increased susceptibility for tuberculosis infection in HTLV-1 infected subjects may be related to impairment in TNF-α production, the severity of tuberculosis in co-infected patients may be due to the enhancement of the Th1 inflammatory response, rather than in their decreased ability to control bacterial growth. The human T cell lymphotropic virus type 1 (HTLV-1) has a worldwide distribution; it is most prevalent in Central and South America, Central Africa and southwestern Japan ,3. MoreoStrongyloides stercoralis the exaggerated Th1 immune response and increased regulatory T cell response decrease the Th2 immune response, which plays a pivotal role in host defense against helminthes [There is evidence that HTLV-1 infection increases severity and susceptibility to strongyloidiasis, scabies and tuberculosis -9. In palminthes . HTLV-1 lminthes ,13-15. Alminthes . Since tlminthes -18 and ilminthes , it has M. tuberculosis in culture. They were admitted because of toxicity to anti-tuberculosis drugs. No difference in the bacillary load was observed in the two groups at admission. The drug sensibility test revealed one M. tuberculosis isolate with multidrug resistance and another with resistance to isoniazid and streptomycin among the cases. In the control group, there was one isolate with resistance to isoniazid, rifampicin and ethambutol and one with resistance to streptomycin. The findings of the chest x-rays were similar in the 2 groups. Cavitation was observed in more than 60% of the patients with or without HTLV-1 infection. Fibrosis and atelectasis occurred in about 50% of the cases and controls. Parenchymal destruction was higher (15.3%) in patients with HTLV-1 infection and tuberculosis than in patients with only tuberculosis. The radiologic findings in patients with HTLV-1 and tuberculosis were not compatible with those observed in patients with T cell impairment. The most common findings were fibrosis, atelectasis, pleural thickening and cavitation. All of them were similar in the two groups but the parenchymal destruction lesions were 3.5 fold higher in patients with HTLV-1 infection and tuberculosis than in patients only with tuberculosis.There was no significant difference in the demographic characteristics  between the 13 patients with tuberculosis and HTLV-1 infection and 25 patients with only tuberculosis. A large proportion of case (54%) and control patients (64%) were underweight (body mass index < 18.4) and the use of alcohol was also similar in both groups. The clinical manifestations, response to TST and the presence and quantification of acid-fast bacilli (AFB) in the sputum in the two groups are shown on Table The therapy for tuberculosis was similar in both groups. The majority of the patients received rifampicin, isoniazid and pyrazinamide. In 33.3% of the cases and in 28% of the controls, patients also received ethambutol. Alternative drugs were given to 15.4% of the cases and to 8% of controls.Figure The main side effects observed were hepatotoxicity and gastric intolerance  observed in, respectively, 38.5% and 23.1% in the cases and in 28% and 36% in the controls (p > 0.05). While death was observed in 2 (15.4%) of the patients with HTLV-1 infection and tuberculosis, none of the patients died in the group only with tuberculosis. Death occurred in one woman and one man. They both had cavitary lung disease, fibrosis, atelectasis and tissue destruction on chest x-rays. Death was due to acute respiratory insufficiency in one case and sepsis in the other.6cells.Regarding HTLV-1 infection, the majority of the cases were HTLV-1 carriers. Four patients had overactive bladder that is considered to be an oligosymptomatic form of HAM. The proviral load was quite variable ranging from 8079 to 499742 copies/10The spontaneous and PPD induced TNF-α and INF-γ production in supernatants of PBMC are shown in Figure The expression of mRNA for IL-12 in unstimulated cultures and the production of IFN-γ in PBMC cultures stimulated with PPD and in cultures with PPD plus exogenous addition of IL-12 are shown in Figure M. tuberculosis infection [in vivo and in vitro. However, while there was decrease in TNF-α production in PPD stimulated cultures, the INF-γ production in unstimulated cultures was higher in co-infected patients than in patient only with tuberculosis. These data together with the observation that the sputum smear conversion was faster in the cases compared to controls, suggests that impairment in innate immune response may contribute to acquisition of tuberculosis, and that severity of the disease may be due to exaggerated inflammatory response observed in co-infected patients.The association between HTLV-1 infection and tuberculosis is widely documented, but little is known about the influence of HTLV-1 on the outcome of nfection ,13-15. IM. tuberculosis[One important limitation of the present study was the characteristics of patients with tuberculosis. The admission for tuberculosis patients is usually due to severity of disease, severe adverse reactions to anti-tuberculosis drugs, history of treatment abandonment and documentation of multidrug-resistant erculosis. In our erculosis.in vivo” and “in vitro” to PPD [M. tuberculosis, not only increases susceptibility but also makes tuberculosis more severe. The results presented here did not show any difference in the frequency of patients who respond to the TST as well as in the size of the induration when the two groups were compared. These data differ from the literature, but the population studied here was quite different from those included in previous studies. In the other studies, data were generated from HTLV-1 infected subjects without documented active tuberculosis or even without history of tuberculosis or vaccination with BCG [While there is good evidence that HTLV-1 infection increases the risk for tuberculosis ,13-15, v” to PPD -19,21, owith BCG -18. HereM. tuberculosis antigen in HTLV-1 infected subjects compared to controls [in vivo observations, the in vitro studies were also performed in HTLV-1 infected patients without history of tuberculosis. However, it is known that abnormalities in T cell or in antigen present cell function occur in HTLV-1 infection [M. tuberculosis mce1 operon mutant cannot modulate immune response and mount an exaggerated inflammatory response associated with control of bacterial multiplication, but survival is decreased due to severe damage of the lung [In vitro studies showed decrease in lymphocyte proliferation and secretion of cytokines upon stimulation with controls ,21. Siminfection . Therefonfection . Here alnfection . In thisthe lung ,26.M. tuberculosis and HTLV-1. TNF-α plays a key role in protection against M. tuberculosis[M. tuberculosis infection [M. tuberculosis may be due to impairment in TNF-α synthesis after exposure to M. tuberculosis, as well as in other component of the immune response not yet determined. However, as IFN-γ production is produced in high levels in HTLV-1 infection and a type 1 immune response to M. tuberculosis antigens is not suppressed, granuloma formation is observed and bacterial growth is controlled. Additionally, as HTLV-1 infection is associated with an exaggerated inflammatory response, there is more tissue damage, which causes severer tuberculosis in HTLV-1 infected subjects.Compared to seronegative controls, expression of IL-12 was lower in both groups and exogenous addition of IL-12 enhanced IFN-γ production in a similar way in tuberculosis patients co-infected with HTLV-1 and in patients with tuberculosis. However, while there was no difference in IFN-γ production among the groups, in PPD stimulated cultures, TNF-α was lower in PPD stimulated cultures of patients co-infected with erculosis,28. Actunfection ,30. In sWhile the increased susceptibility of HTLV-1 infected patients to acquire tuberculosis may be due to impaired TNF-α production, the severity of tuberculosis in HTLV-1 infected individuals may be related to an increased inflammatory response.The study participants included 13 patients with diagnosis of tuberculosis and HTLV-1 infection and 25 patients who had tuberculosis without HTLV-1. All patients with tuberculosis consecutively admitted to Hospital Especializado Octávio Mangabeira (HEOM), Salvador, Brazil, from August 2007 to July 2009 were tested for HTLV-1 by ELISA and those who tested positive underwent confirmation by a Western blot test. The inclusion criteria were age between 18 to 60 years, diagnosis of tuberculosis with documentation of acid-fast bacilli (AFB) by microscopy in the sputum and serologic test for HTLV-1. Exclusion criteria included pregnant women, patients with diabetes, kidney failure, HIV infection and use of immunosuppressive drugs. For each patient with tuberculosis and HTLV-1 infection, two controls with only tuberculosis matched by gender and age plus or minus five years of age were included. A case was defined as a patient with two sputum tests positive for AFB by microscopy and a positive ELISA test for HTLV-1 confirmed by Western blot. A standardized questionnaire soliciting demographic and clinical information was completed for each participant at the time of enrolment into the study. A pulmonary specialist (MLB) examined each patient and reviewed laboratory test and the x-ray results. The patients were evaluated at entrance into the study, and at 10, 20 and 30 days after admission. An informed consent was obtained from all participants and the study was approved by the Ethical Committee of the HEOM.The TST was performed by trained nursing staff at HEOM with 0.1 ml of PPD RT23 . The reaction was read 72 h later. The cut-off point for a positive reaction was > 5 mm induration.Serum samples were screened for antibodies to HTLV-1 by ELISA  and confirmed by a Western blot analysis  performed in accordance with the manufaturer’s instructions. Samples were also screened for antibodies to HIV-1 by ELISA . Sputum of each patient was examined by the Ziehl-Neelsen staining method and the bacillary load was classified as 1+, 2+, 3+ or negative. Culture of the sputum was also performed in Lowenstein-Jensen culture media.6ml and cultured in RPMI 1640  plus 5% heat inactivated human AB serum supplemented with gentamicin and glutamine. Cells were incubated only with medium or stimulated with PPD at concentration of 1μg/ml for 72 hours at 37°C in 5% CO2. Determination of TNF-α, IFN-γ and IL-10 was performed by ELISA with monoclonal anti cytokine antibodies [TM Software v2.0 (AB Applied Biosystems). Data for stimulated cultures represent the production of cytokine obtained in culture stimulated with PPD. In some experiments IL-12 and anti-IL-10 were added to the cultures and IFN-γ was measured in supernatants.Peripheral blood mononuclear cells (PBMC) were obtained from heparinized venous blood by density gradient centrifugation using Ficoll-Hypaque . After washing with saline, the cells were adjusted to 3x10tibodies . BrieflyThe HTLV-1 proviral load was performed after DNA extraction from mononuclear cells as previously described .t-test for continuous variables and Pearson x2 test for categorical variables. Fisher’s exact test was used when one the comparison groups had an expected frequency of five or less. Kaplan-Meier survival analysis was used to compare sputum microscopy conversion after 10 and 20 days of tuberculosis treatment. Comparison of TNF-α, IL-12 and IFN-γ production between the groups was performed by the Mann Whitney U Test. All analysis was performed with statistical software STATA 10.1 .Hospitalized pulmonary TB patients with HTLV-1 were compared to control patients without HTLV-1 by Student The authors declare that they have no competing interests.MLB was responsible for the clinical evaluation of the patients, X-ray analysis, data analysis and preparation of the manuscript. OB, TB, IC, IB, FB and DP assisted MLB in the clinical evaluation and followed the patients during hospitalization. BF and LR performed the statistical analysis and review of the manuscript. SS, AS and EMC were responsible for immunological studies. MLB, EMC and LR also participated of the study design, data analysis and in the preparation of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/199/prepub


Patients with HTLV-1 associated myelopathy (HAM/TSP) tend to present higher HTLV-1 proviral load (PVL) than asymptomatic carriers (AC). PVL remains the only marker consistently associated with HAM/TSP across several populations. We report a candidate gene study to identify associations between host genetic markers and PVL.170 HTLV-1 positive individuals were analyzed (56 HAM/TSP and 114 AC). Six HLA alleles and 140 SNPs belonging to 40 candidate genes were genotyped, with 36 ancestry informative markers included into the analysis to correct by population stratification. PVL was determined by a SYBR Green-based realtime quantitative PCR using ERV-3 as reference gene.The effect of each SNP was evaluated with linear regression analysis. Disease status, age, gender, and the first three principal components were included as covariates.Due to the exploratory nature of the study, no correction for multiple testing was performed; a P value<0.05 was considered significant.After multivariate linear regression analysis, two SNPs: IL6–174 (Interleukine 6) (P=0.0013) and ICAM1-R241G (Intercellular Adhesion Molecule 1) (P=0.0084), and HLA-A*02 (P=0.0306) showed association with PVL.These results suggest a possible implication of IL6–174, ICAM1-R241G and HLA-A*02 allele in the PVL among Peruvian HTLV-1 infected individuals. IL-6 promotes T cell activation, growth, and differentiation of cytotoxic T lymphocytes; ICAM-1 was reported as cofactor for HTLV-1-induced cell fusion and is upregulated in HTLV-1 infected cells; a protective effect associated with TAX has been suggested for HLA-A*02. A replication study in an independent population is needed to confirm our findings.


Hepatozoon infection and compared its performance with microscopy and PCR. Using qPCR, we also compared various protocols that differ in the biological source and the extraction methods. Our results show that the qPCR approach on DNA extracted from blood samples, regardless of the extraction protocol, provided the most sensitive estimates of Hepatozoon infection parameters; while allowed us to differentiate between mixed infections of Adeleorinid (Hepatozoon) and Eimeriorinid (Schellackia and Lankesterella), based on the analysis of melting curves. We also show that tissue and saline methods can be used as low-cost alternatives in parasitological studies. The next step was to test our qPCR assay in a biological context, and for this purpose we investigated infection patterns between two sympatric lacertid species, which are naturally infected with apicomplexan hemoparasites, such as the genera Schellackia (Eimeriorina) and Hepatozoon (Adeleorina). From a biological standpoint, we found a positive correlation between Hepatozoon intensity of infection and host body size within each host species, being significantly higher in males, and higher in the smaller sized host species. These variations can be associated with a number of host intrinsic factors, like hormonal and immunological traits, that require further investigation. Our findings are relevant as they pinpoint the importance of accounting for methodological issues to better estimate infection in parasitological studies, and illustrate how between-host factors can influence parasite distributions in sympatric natural populations.Identifying factors influencing infection patterns among hosts is critical for our understanding of the evolution and impact of parasitism in natural populations. However, the correct estimation of infection parameters depends on the performance of detection and quantification methods. In this study, we designed a quantitative PCR (qPCR) assay targeting the 18 S rRNA gene to estimate prevalence and intensity of  Parasites can be major drivers of host ecology and evolution and play key roles in ecosystem functioning and structure. Parasitism can for example alter host life-history traits and fitness Importantly, the estimation of biological relevant and realistic infection patterns is constrained by differences in the accuracy, specificity and sensitivity of the detection and quantification protocols used Sympatric and closely related species represent natural model systems and an ideal opportunity where to investigate between-host differences in infection parameters Hepatozoon (Apicomplexa: Adeleorina), is a highly diverse genus of intracellular hemogregarine parasites with more than 300 species described Hepatozoon infections in reptiles is unclear, with studies reporting from apparently non-detrimental infections in natural hosts Schellackia sp. (Apicomplexa: Eimeriorina), are often found at lower prevalence and intensities in wild reptile hosts, and mixed infections with Hepatozoon can also occur Hepatozoon infection parameters in two sympatric closely related lacertid species, Podarcis bocagei and P. hispanica. These two host species present an attractive system because: first, they live in sympatry and, despite having similar ecological requirements, there are apparent preferences for microhabitat: P. hispanica is frequently found on rocks (saxicolous), while P. bocagei is mainly grounddwelling P. bocagei being generally larger Hepatozoon and low prevalence of Schellackia infections for these species in the Iberian Peninsula Hepatozoon infection, while controlling for confounding factors such as ecology and host and parasite phylogeny.Hepatozoon infection in two sympatric closely related lacertid species, P. bocagei and P. hispanica, and assess the relative role of inter-individual (intra-species) and between-species factors on these parameters.The objectives of this study are: i) to evaluate the accuracy and sensitivity of different detection methods  and protocols  for detecting and quantifying hemogregarine parasites in reptile samples, as well as the occurrence of mixed infections, and ii) apply the most sensitive approach, i.e. qPCR, to determine prevalence and intensity of P. bocagei and P. hispanica, from a single location in Gerês, Northern Portugal , in July 2011. Capture permits were issued by the ICNB , license numbers 67-75/2011/CAPT. These hosts are small, diurnal, insectivorous lizards, with adult snout-vent length (SVL) of 45–65 mm and 37–70, respectively. A total of 87 adult individuals were sampled . Several photographs per slide were taken under the 40× magnification lenses and stitched using cell∧B software (basic image-acquisition and archiving software). Prevalence was estimated as the proportion of infected hosts and intensity of infection was estimated as the number of parasites per 4,000 erythrocytes from a total of 72 blood smears n = 87), DNA was extracted from blood using the Speedtools tissue DNA extraction kit . In a subset of 47 individuals, in which we compared various methodological approaches, DNA was extracted from two biological sources: blood drops stored in Whatman filter paper (approximately 3 mm by 3 mm) and tail-tip muscle tissue (approximately 2 mm by 2 mm with skin removed). On these samples we used two extraction protocols: the Speedtools commercial kit following manufacturer's instructions, and the standard saline protocol For the total number of samples (Hepatozoon 18 S rRNA gene. The PCR reactions using the Hep primers were run in a 20 µl reaction mixture containing 1 U of GoTaq DNA Polymerase (5 u/µl), 1.5 mM MgCl2 (25 mM), 0.125 mM of each nucleotide, 1 X GoTaq Flexi Buffer, 0.6 mM of each primer, and 2 µl of DNA. The reaction mix was heated to 94°C for 3 min, and amplification was performed through at 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min, in 35 cycles, with a final 10 min extension at 72°C. Two negatives (one blank and one known Hepatozoon negative) and one positive control (one known Hepatozoon positive) were run with each reaction. Sequencing of the positive PCR products was performed in both directions outsource . Geneious v6.0.3 was used for contig assembly and visualization of sequences. The “heterozygotes plugin” with 30% peak similarity was used to search for possible heterozygous positions. Consensus sequences for each individual were deposited in GenBank under the accession numbers KJ189387-KJ189433 (Hepatozoon), KJ189382-KJ189385 (Schellackia) and KJ189386 (Lankesterella). One representative of each of the three Hepatozoon haplotypes , of the two Schellackia haplotypes (KJ189382 and KJ189384), and the single Lankesterella haplotype (KJ189386), were aligned with published data on GenBank using the MUSCLE alignment 6 generations, saving one tree with random tree sampling each 1000 generations. All trees prior to reaching stationarity (25% of the run) were discarded as burn-in samples, and the remaining trees were combined in a 50% majority consensus tree.PCR amplifications were performed on all 87 samples using primers HepF300 and HepR900 Hepatozoon species in canids have been previously designed Hepatozoon and other apicomplexans (5′-ACTCACCAGGTCCAGACATAGA-3′) and JM5_R (5′-CTCAAACTTCCTTGCGTTAGAC-3′) using Primer3Plus Hepatozoon 18 S rDNA fragment (171 bp) amplified from a positive P. hispanica individual (JX531917) that we used as amplification standard for the qPCR assay. The PCR product was cloned to a plasmid using the TOPO TA Cloning kit  following the manufacturer's instructions. One Shot TOP10 Competent Cells were used for transformation and we used the Purelink Quick Plasmid Miniprep Kit  to isolate the plasmid . Successful plasmid integration of the Hepatozoon gene fragment was assessed using M13 primers following manufacturer's instructions. Serial 10-fold dilutions containing from 500,000 to 5 copies of plasmid were performed to produce a standard curve for Hepatozoon quantification that was included in each qPCR plate. A MyiQ qPCR machine (Applied Biosystems) was used with the following protocol: 95°C 10 min, 95°C 10 s, 63°C 20 s, 72°C 25 s (with melting), 95°C 1 min, 63°C 30 s, melting from 63–95°C by increments of 0.2°C every 10 s. Reactions contained iQ SYBR Green supermix at 0.5x, each primer at 0.5 mM, 2 µl of 10 ng/µl genomic DNA, in a total volume mix of 20 µl. To estimate the number of copies in unknown samples, raw qPCR results were exported using the program iQ5 R&D version 2.1 (Biorad) and the baseline threshold was determined individually for each plate using the algorithm implemented in LinRegPCR Hepatozoon from microscopy and PCR) and all those displaying mixed infection peaks. These sequences have been deposited in GenBank under accession numbers KJ189434-KJ189463. For this experiment, samples with all threshold cycles (CTs) higher than 35 were considered negative because repeatability decreased significantly after cycle 35, with most replicates differing by more than one Ct. Mixed infection intensity estimates (n = 6) were discarded from subsequent analyses.qPCR assays for detecting mplexans . For thiHepatozoon copy number) were log-transformed using the formula log (x+1). Fitness of the models was tested by Normal Q-Q plots of the Pearson residuals from the model analysis.Normality tests were performed using Shapiro-Wilk and homogeneity of variances tested using Levene's non-parametric test. To approach normality, infection estimates obtained from qPCR . We used McNemar's test to compare sensitivity of the three detection methods (n = 87) and the four extraction protocols (n = 47) in estimating Hepatozoon prevalence. Then, to compare Hepatozoon intensity among biological sources and extraction protocols  we fitted a Generalized Linear Mixed Model (GZLMM), as recommended for unbalanced data with repeated measures (random effects) We first investigated the correlation between Podarcis host species and between sexes within each species. Prevalence of Hepatozoon infection between host species, between sexes within each species and between the same sexes of both species (n = 87) was compared using a Chi-square test (http://www.quantpsy.org/chisq/chisq.htm). For intensity comparisons between host species and sexes , while accounting for the effects of body size, we used a Generalized Linear Model (GZLM) with normal distribution and log link function using Type I SS. The full model included body size, host species and sexes (and their interactions) as fixed factors.Previous studies have found various associations between infection parameters and body size All statistical analyses were conducted in IBM SPSS Statistics 21, except for McNemar tests that were conducted in R .Hepatozoon infection estimated by microscopy and qPCR were significantly correlated , with qPCR being much more sensitive and detecting as few as 5 copies of parasite DNA. Mean threshold cycle (Ct) standard deviation (SD) was 0.16, mean efficiency 81.1% (SD±2.6) and mean R2 = 0.991 (SD±0.008)  presented a mixed electropherogram in the sequencing analyses. These mixed infections detected by qPCR matched those detected by conventional PCR sequencing.Intensity of D±0.008) . Mean SDHepatozoon spp. (Apicomplexa: Adeleorina), 4 with Schellackia spp. and 1 with Lankesterella spp. (Apicomplexa: Eimeriorina). These were considered positive for PCR prevalence  resulted in 3 unique haplotypes , and the other haplotype (two sequences from P. hispanica and one from P. bocagei) was closely related with two haplotypes from Lacerta schreiberi (JX984674 and JX984675) (Lankesterella sp. sequence obtained in this study is related with Lankesterella minima from the American bullfrog Lithobates (Rana) catesbeianus (AF080611) and Lankesterella valsainensis from the blue tit Parus caeruleus (DQ390207)  . FinallyQ390207) .Hepatozoon prevalence, including microscopy, PCR and qPCR  and PCR . Microscopy and PCR also differed significantly . Among molecular methods: 23% (16/69) of qPCR Hepatozoon positives and 75% (3/4) of qPCR Eimeriorina positives were not detected by PCR, while the contrary was found only for 2% (1/51) Hepatozoon and 20% (1/5) Eimeriorinid positives (Hepatozoon negatives for PCR and qPCR (considering only blood and the kit extraction protocol) were also negative by microscopy, but two Eimeriorina positives by microscopy were not detected by PCR , but not for the type of extraction protocol . In terms of Hepatozoon intensity of infection, the GZLMM analysis showed that Hepatozoon intensity differed significantly between sources  (P = 0.035), but their interaction was marginally significant . Pairwise comparisons further show that the two tissue extractions differ significantly  (P = 0.830)  and two extraction protocols  in estimating ividuals . PrevaleP<0.001)  and betw= 0.027) , but not= 0.830) , which s the kit . Overallntensity  and provntensity . In addintensity . Finallyntensity .P = 0.001), P. bocagei being bigger, and sexes , males being bigger in size. Given that qPCR and blood-kit DNA extraction provided good estimates of infection parameters for the methodological subset comparison, we used this protocol for investigating the relationship between host-factors and infection patterns. Prevalence was higher in P. hispanica females (89%) than in P. bocagei females (59%) , but there were no significant differences between species , or sexes    and sexes , on Hepatozoon intensity levels, and confirmed a significant effect of the covariate factor body size . The interaction between body size and host species was also significant , which indicates that the effect of host species on the intensity of infection is body-size dependent  see . In term= 0.014) . The GZLependent .Hepatozoon prevalence and intensity, especially in cases of low intensity levels, which is concordant with previous studies conducted on PlasmodiumHepatozoon parasites of reptiles, with the few qPCR studies on Hepatozoon focusing on carnivore hepatozoonosis Hepatozoon prevalence and intensity levels, regardless of the extraction protocol, and should therefore be the preferred collection method during fieldwork when possible. Our results also show that the traditional saline DNA extraction protocol can be used as an effective low-cost alternative to the tested commercial DNA extraction kit, since it provides similar estimations of hemoparasite infections. This result is in agreement with previous parasitological studies using alternative protocols to commercial kits Hepatozoon infection intensity from tissue was proportionally lower than blood, probably because a piece of tail-tip muscle contains less DNA, than a blood drop stored in Whatman filter paper. It is possible that increasing the DNA concentration of tissue extractions used in qPCR would yield similar results to blood, but this was not tested in this study. The same applies to tissue extracted with kit and tissue extracted with the saline protocol, the latter providing lower overall estimates of Hepatozoon infection. Thus, we recommend the use of higher DNA concentrations when using tissue samples. Our results highlight the need of comparing multiple methods in a case-base manner as a preliminary step, in order to minimize imperfect detection that can ultimately lead to erroneous ecological and epidemiological inferences In this study we compared the performance of various detection methods and DNA extraction protocols, and evaluated how the detection method can constrain the estimation of biological relevant infection parameters. First, by comparing various methodological approaches, we found that qPCR was the most accurate and sensitive method for estimating Hepatozoon (Adeleorina) with two genera of Eimeriorinid parasites (Schellackia and Lankesterella). These parasites were detected by the three detection methods , but with different sensitivities. Conventional PCR using the Hep primers only allow the amplification of either hemogregarines or other apicomplexans, and preferentially amplify non-hemogregarine parasites when present in mixed infections Schellackia positives detected by PCR. The false Eimeriorina negative of the qPCR approach could be due to handling errors (e.g. pipetting) or, most likely, because of the preferential binding of the qPCR primers towards Hepatozoon. Despite the qPCR melting curve analysis allowed us to differentiate between parasite genera, specificity was not high enough to discriminate at lower levels, i.e. between closely related Hepatozoon haplotypes. This result is not unexpected, as the aim of this study was to simultaneously detect and quantify various hemogregarine parasites in reptile samples with high accuracy and sensitivity, so the primer set used herein targets an invariable region of the 18 S rRNA gene (see P. hispanicaSchellackia and Lankesterella parasites (Apicomplexa: Lankesterellidae). The fact that they are present in both Podarcis species at low prevalence and low intensities may be an indication of the opportunistic nature of the interaction, and/or the fact that this parasite may have detrimental effects on hosts. Co-infection implications in wildlife hosts remain poorly investigated but interaction among parasite heterospecifics in mixed infections is often asymmetrical In this study we found mixed infection of gene see . Thus, fHepatozoon infection parameters in two Podarcis sympatric species. Our results show high levels of prevalence with no significant differences between host species and sexes within each species. Prevalence can be determined by the rate at which parasites encounter suitable hosts Based on qPCR estimates (from blood-kit DNA extraction) we investigated Hepatozoon intensity of infection, we found that it was positively associated with body size within each host species, which seems to be a common pattern among short-lived reptile species (e.g. Iberolacerta monticolaP. lilfordiLacerta viridisRegarding P. hispanica) harboured significantly higher intensity levels of Hepatozoon parasites. The association between body size and intensity of infection differed for the two species, being more pronounced in P. bocagei, for which a lower proportion of smaller animals and a higher proportion of bigger animals, are infected. Also, males had higher intensities than females. Several factors may explain these patterns including: host immunocompetence and susceptibility to infection P. hispanica, and males within each species, can be more susceptible to infection and/or can differ immunologically/hormonally, and thus harbour higher hemogregarine intensity levels. Differences in leukocyte counts have been reported between Podarcis species When comparing intensity levels between the two species, the smaller sized species Click here for additional data file.Figure S2Standard curve obtained from a six 10-fold serial dilutions of the plasmid containing the 18 S rRNA gene. Numbers correspond to dilutions from 1  to 6 (5 copies). Dashed lines indicate the number of copies for an unknown sample determined based on the starting Threshold cycle (Ct).(TIF)Click here for additional data file.Table S1Number of haplotypes and heterozygous individuals found in this study. The symbol + indicates when the first haplotype peaks are higher, while & indicates when both haplotype peaks are approximately of the same height.(DOC)Click here for additional data file.


Human T-cell leukemia virus type 1 (HTLV-1) is associated with two immunologically distinct diseases: HTLV-1-associated myelopathy/tropical spastic paraparesis and adult T-cell leukemia. We observed previously that depletion of dendritic cells (DCs) in CD11c-DTR transgenic mice followed by infection with cell-free virus led to greater proviral and Tax mRNA loads and diminished the cellular immune response compared to mice infected with cell-associated virus. To understand the significance of these in vivo results and explore the host pathogen interaction between dendritic cells and cell-free HTLV-1, we used Fms-like tyrosine kinase 3 ligand (Flt3L) cultured mouse bone marrow-derived DCs (FL-DCs) and chimeric HTLV-1. Phenotypically, the FL-DCs upregulated expression of surface markers  on infection; however, the level of MHC class I remained unchanged. We performed kinetic studies to understand viral entry, proviral integration, and expression of the viral protein Tax. Multiplex cytokine profiling revealed production of an array of proinflammatory cytokines and type 1 IFN (IFN-α) by FL-DCs treated with virus. Virus-matured FL-DCs stimulated proliferation of autologous CD3+ T cells as shown by intracellular nuclear Ki67 staining and produced IFN-γ when cultured with infected FL-DCs. Gene expression studies using type 1 IFN-specific and DC-specific arrays revealed upregulation of interferon-stimulated genes, most cytokines, and transcription factors but a distinct downregulation of many chemokines. Overall, these results highlight the critical early responses generated by FL-DCs on challenge with cell-free chimeric HTLV-1.


Singly spliced mRNA from orf-I encodes p12, which can be proteolytically cleaved to generate p8, while differential splicing of mRNA from orf-II results in production of p13 and p30. These proteins have been demonstrated to modulate transcription, apoptosis, host cell activation and proliferation, virus infectivity and transmission, and host immune responses. Though these proteins are not essential for virus replication in vitro, p8, p12, p13, and p30 have an important role in the establishment and maintenance of HTLV-1 infection in vivo.The 3′ end of the human T-cell leukemia/lymphoma virus type-1 (HTLV-1) genome contains four overlapping open reading frames (ORF) that encode regulatory proteins. Here, we review current knowledge of HTLV-1  I. I116]. 4.6.orf-II peptides can be detected in HTLV-1-infected individuals, suggesting that p13 may have an important role in vivo [in vivo, a molecular clone of HTLV-1 mutated to selectively ablate p13 failed to establish viral infection in a rabbit model [CTLs and antibodies that recognize  in vivo ,117. In it model . Howeverit model . No stud5.In vivo studies demonstrate that HTLV-1 requires orf-I and orf-II for viral persistence. Since these proteins play critical roles in T-cell activation, MHC-I trafficking, cytokine expression, and virus replication, they are likely crucial for HTLV-1 maintenance of low level of virus expression in vivo. Together, p12 and p8 decrease ICAM-1, ICAM-2, and MHC-I expression at the cell surface, limiting the ability of NK cells and CTL cells to recognize infected cells. In addition, p8 inhibits proximal TCR signaling upon TCR stimulation to prevent T-cell activation. In contrast, within the ER, p12 promotes Ca2+ release and NFAT activation in resting cells. Thus, p12 decreases the IL-2 requirement for proliferation in the presence of suboptimal antigen presentation. Altogether, these data suggest that the concerted expression of p12 and p8 modulate T-cell activation and antigen presentation while promoting proliferation of resting cells, therefore providing new target cells for the virus. Orf-II protects HTLV-1-infected cells from immune system recognition by dampening the transcriptional effects of Tax. In the nucleus, p30 binds and prevents export of tax/rex mRNA to the cytosol for translation and p13, upon ubiquitination, inhibits Tax-CBP/p300 complex formation. This results in a decrease in the transcriptional activity of Tax and subsequent virus replication. In macrophages, p30 favors the production of anti-inflammatory IL-10 while decreasing the expression of pro-inflammatory cytokines by interacting with the transcription factor PU.1. Furthermore, like orf-I, p30 and p13 promote proliferation of resting T-cells. p30 inhibits DNA repair mechanisms to favor the growth and persistence of infected cells. p13 modulates the production of ROS and proliferation of resting cells while promoting apoptosis of transformed cells. The understanding of how these proteins simultaneously protect HTLV-1-infected cells from destruction and promote their expansion leads to new questions. Where, when, and at what level these proteins are expressed in vivo still must be elucidated. The answers to these questions will indicate whether these proteins may provide new targets for HTLV-1 therapeutics.HTLV-1-associated diseases have long periods of clinical latency with infected individuals having life-long persistence of viral-infected T-cell clones. Thus, HTLV-1-infected T-cells must be able to avoid immune recognition. As transcription of the provirus could lead to antigen presentation and immune recognition, it is necessary for HTLV-1 to maintain low levels of virus replication.


Hepatozoon spp. infection in Mashhad, northeast of Iran, via blood smear parasitology.We attempted to determine the prevalence of The prevalence was investigated by examination of blood smear parasitology, using blood samples collected from 254 dogs (51 strays and 203 privately owned-dogs).Hepatozoon spp. Therefore, as per blood smear parasitology, the prevalence of Hepatozoon spp. infection was 1.57% (4/254). Sixteen out of 254 dogs (6.29%) were infested with ticks; all of which were Rhipicephalus sanguineus. One of the dogs infected with Hepatozoon spp. exhibited ticks at the time of examination. Concurrent infection with Ehrlichia canis and Leishmania infantum was not detected in the four Hepatozoon spp. infected dogs.Two stray dogs  and two privately-owned dogs  were infected with Hepatozoon spp. infection in dogs in Iran.This is the first epidemiological study on the prevalence of  Hepatozoon is a tick borne protozoan parasite, classified in the Phylum Apicomplexa and is closely related to Plasmodium spp. and Piroplasms  were drawn from the cephalic vein of each dog and were placed into plain and Ethylenediaminetetraacetic acid (EDTA) tubes. A thin blood smear was prepared for each sample, fixed with methanol and stained with Giemsa. Complete blood counts were performed manually for all dogs, and the presence of hematological disorders was recorded for each animal. The parasitemia was calculated manually by counting 500 neutrophils under oil immersion field  test kit  in accordance with the manufacture's recommendations. The stained slides were read under 400 magnification fluorescence microscope . Evaluation was carried out with green and red filter.The sera were separated off and frozen at 20 °C for serological assays. All sera were examined for the presence of the antibodies against Hepatozoon spp.  of the subjects were infected by oon spp. . These iR. sanguineus. The ticks were found at the surface of the skin on one of the four infected dogs.Out of 254 dogs, 16 (6.29%) were infested with ticks; all of which were Hepatozoon spp. infected. Hepatozoon spp. infected dogs.Anorexia was observed in two infected dogs. Anemia was detected in 67 of the 254 dogs (26.37%), two of which were L. infantum. Also, a very low prevalence of anti-E. canis antibodies , was detected among studied dogs (unpublished data). In the four Hepatozoon spp. infected dogs, concurrent infection with these pathogens were not detected.Twenty-two of 254 (8.67%) serum samples had antibodies to Hepatozoon spp. infection among dogs living in Mashhad, northeast of Iran. As per this study, the prevalence of Hepatozoon spp. infection was 1.57% by examination of blood smear parasitology. In contrast to the present study, others have reported a rather high prevalence of Hepatozoon spp. infection. Garcia de Sá et al. examined 31 dogs from rural areas and identified 7 dogs (22.6%) positive by blood smear examination  and the other three were young (1.5 to 3 yr). Hepatozoon spp. infection was found in dogs from 9 months to 7 years old , developed the severe form of the disease. Previously, Khoshnegah et al. described a severe form of hepatozoonosis occurred in an 11-year-old male dog. Clinical signs included anorexia, weight loss, depression, nasal and ocular discharge, in coordination of the posterior limbs, a mildly painful hind limb, peripheral lymphadenopathy, pale mucous membranes but afebrile  were not detected in the four Hepatozoon spp. infected dogs. There is also the possibility that an interaction between the different pathogens might exist, which in turn might lead to further deterioration in the infected dogs’ clinical condition (In the present study, the infected dogs were asymptomatic, with low parasitemia, ranged from 1% to 3%. A low level of al signs . The preafebrile . In casenia, etc –23. In tondition .Hepatozoon spp. infection among dogs residing in the northeast of Iran. One limitation of the direct observation is that gametocytes are not always detectable since parasitemia may be intermittent or the number of circulating gametocytes very low. From a clinical perspective, practitioners should be aware of Hepatozoon spp. and should include its screening in their routine diagnostic panel. Misdiagnosis may lead to inappropriate treatment and relapses. As soon as the disease is diagnosed, practitioners should know that since elimination of gametocytes from the peripheral blood is slow, an 8-week treatment is always required (H. canis or by a new species of Hepatozoon and could be considered endemic. Further work based on PCR and serological tests is necessary to confirm the species causing this disease in Iran. Most importantly, strict control measures against ticks applied in dog kennels and shelters may prevent the spread of tick-borne diseases.The present study demonstrates a low prevalence of required . Canine


Human T-cell leukemia virus type-1 (HTLV-1) infection causes adult T-cell leukemia (ATL). Recently, a novel viral protein HTLV-1 bZIP factor (HBZ), which is encoded an antisense viral gene, has been identified. HBZ may have a functional key player in cellular leukemogenesis, but the function in cells is poorly understood. To characterize the function(s) of HBZ, we performed yeast two-hybrid screen using HBZ as bait and identified growth arrest and DNA damage gene 34 (GADD34). GADD34 is induced by endoplasmic reticulum (ER) stress and the impairment of DNA. Both endogenous HBZ and GADD34 could interact in HTLV-1 infected cells. HBZ interacts with C-terminal region of GADD34 via its N-terminal region in mammalian cells. HBZ and GADD34 showed the same subcellular localization. GADD34 promotes the dephosphorylation of the factors which are downstream of mammalian target of rapamycin (mTOR), therefore, GADD34 represses mTOR activity. Our current study is focused on understanding the molecular mechanisms by which the interaction between GADD34 and HBZ regulates the mTOR signaling pathway.


Human T-cell Lymphotropic Virus type-1 (HTLV-1) is the causative agent of an aggressive malignancy of CD4+ T lymphocytes. It is believed that the viral transactivator Tax-1 is a major player in T-cell transformation. Thus, targeting Tax-1 protein is regarded as a possible strategy to arrest viral replication and ultimately to counteract neoplastic transformation.Here, we demonstrated that CIITA, the master regulator of MHC class II gene transcription, inhibits HTLV-1 replication by blocking the transactivating function of Tax-1. Co-immunoprecipitation experiments have shown that CIITA and Tax-1 physically interact in vivo and that the first 108 amino acids of Tax-1 were necessary for this binding. Two adjacent regions (1-252 and 253-410) of CIITA bound independently to Tax-1, but only region 1-252 mediated Tax-1 inhibition, in agreement with the fact that CIITA residues from positions 64-124 were required to block Tax-1 transactivation. CIITA inhibitory action on Tax-1 function correlated with the nuclear localization of CIITA and was independent of the transcription factor NF-YB, previously involved in CIITA-mediated inhibition of Tax-2 of HTLV-2, a virus with still elusive pathogenic action. Furthermore, CIITA severely impaired the physical and functional interaction of Tax-1 with the cellular co-activator PCAF, which is required for the optimal activation of HTLV-1 promoter. Accordingly, the over-expression of PCAF restored Tax-1-dependent transactivation of the viral LTR promoter inhibited by CIITA.These findings strongly support our original observation that CIITA, beside increasing the antigen-presenting function for pathogen antigens, acts as an endogenous restriction factor against human retroviruses by blocking virus replication and spreading.


Airway remodeling due to increased airway smooth muscle (ASM) mass, likely due to enhanced migration and proliferation, has been shown to be highly associated with decline in lung function in asthma. Thymic stromal lymphopoietin (TSLP) is an IL-7-like, pro-allergic cytokine that has been shown to be necessary and sufficient for the development of allergic asthma. Human ASM (HASM) cells express TSLP receptor (TSLPR), the activation of which leads to enhanced release of proinflammatory mediators such as IL-6, CCL11/eotaxin-1, and CXCL8/IL-8. We show here that TSLP induces HASM cell migration through STAT3 activation since lentiviral-shRNA inhibition of STAT3 abrogated the TSLP-induced cell migration. Moreover, TSLP induced multiple cytoskeleton changes in HASM cells such as actin polymerization, cell polarization, and activation of small GTPase Rac1. Collectively, our data suggest a pro-migratory function of TSLP in ASM remodeling and provides better rationale for targeting TSLP/TSLPR pathway for therapeutic approaches in allergic asthma. Airway remodeling is a cardinal feature in allergic asthma that culminates in the form of abnormal epithelium, subepithelial thickening, mucus gland hypertrophy, alteration in extracellular matrix deposition, and increase in airway smooth muscle (ASM) mass. ASM remodeling has been shown to be one of the most crucial factors that correlate with decreased lung function in asthma2+712Thymic stromal lymphopoietin (TSLP) is a pro-allergic, IL-7-like hematopoietic cytokine that has been shown to be necessary for the development of allergic asthma in some animal modelsTo assess whether TSLP can affect HASM cell migration, Boyden chamber assay for cell migration was performed on primary HASM cells. We found that the recombinant human TSLP (1–10 ng/ml) induces HASM cell migration . As shown in We then sought to investigate the underlying signaling mechanisms of TSLP-induced cell migration. We have previously established that Signal transducer and activator of transcription 3 (STAT3) but not STAT5 is phosphorylated and only STAT3 activation is required for TSLP-induced HASM cell synthetic functionsMethods section). As shown in representative micrographs of phalloidin-stained cells in Reorganization of actin cytoskeleton is a fundamental mechanism in cell motility and migration. To further unravel the potential mechanisms of actin substructures involved in migration process, we employed cytoskeleton staining of HASM cells by using phalloidin-Alexa Fluor® 488  (See Methods section). As shown in The primary requirement for lamellipodia formation is the activation of small guanosine triphosphate hydrolase (GTPase) Rac1 within minutes of extracellular stimuliThe data presented here uniquely shows a pro-migratory function of TSLP in HASM cells. TSLP-induced HASM cell migration was dependent upon STAT3 activity as shown by lentiviral-shRNA-mediated STAT3 inhibition experiments. Further analysis showed that TSLP invokes necessary cytoskeleton rearrangements such as formation of lamellipodia, cell polarization, and enhanced activity of small GTPase Rac1. Collectively, we show that TSLP may induce pro-migratory function in human ASM cells.N. S. Redhu, L. Shan, and A. S. Gounni, unpublished observations). Therefore, this data provides a strong rationale for further studies in animal models to assess the role of TSLP in airway smooth muscle remodeling. In support of our proposal, a recent study using house dust mite (HDM) allergen-induced asthma model showed that neutralization of TSLP with anti-TSLP mAb reversed the airway inflammation, prevented structural alterations, and decreased the pro-remodeling cytokine TGF-β expression and AHR to methacholineMigration of smooth muscle occurs during tube formation of hollow organs including blood vessels, gastrointestinal tract, and airwaysAlthough TSLP has recently emerged as a direct player in initiation of allergic inflammatory responses, it was initially recognized as a growth factor and mitogen for pro-B cell line Ba/F310112314The process of cell migration is initiated by activation of receptors such as G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTK), and integrins which trigger the remodeling of cytoskeleton, discussed in context of HASM cell migration extensively inTaken together, as the appreciation for the role of TSLP in initiation and/or perpetuation of airway inflammation is growing, current report underscores a potential pro-remodeling function of TSLP. TSLP-induced HASM cell migration involves STAT3 activity, actin polymerization, cell polarization, and Rac1 activity. It is highly likely that locally produced TSLP in airways , besides activating the DCs to shape the inflammatory Th2 differentiation, can act in an autocrine/paracrine manner to directly induce local ASM and epithelial tissue remodeling, prominent in allergic asthma. Therefore, strategies targeting the TSLP, its receptor, or signaling components may additionally be considered for trials in airway remodeling.Recombinant human TSLP and PDGF-BB were purchased from R&D Systems . FBS was from HyClone Laboratories . DMEM, Ham's F12, trypsin-EDTA, antibiotics , were from Invitrogen Life Technologies . All other reagents were procured from Sigma-Aldrich Canada Ltd. , unless specified.Three different sources of HASM cells were used. Written informed consent was obtained from the tissue donors, and this study was approved by the research ethics committee of the University of Manitoba, Winnipeg, Canada. Both hTERT-immortalized and primary human bronchial smooth muscle (HBSM) cells were prepared as described previously1231324 cells/ml) was added to the upper chamber of modified Boyden chamber apparatus (Neuroprobe). In the lower chamber, TSLP  or PDGF-BB (10 ng/ml) were added as chemoattractant to the same media as the upper chamber. After 4 h of incubation at 37°C in humidified 5% CO2 incubator, the membranes were peeled-off. Cells on the upper side of the membrane were scraped off and the cells migrated to the lower side were fixed and stained with Hemacolor® stain set . The number of migrated cells was counted in four-five random fields under X20 magnification by phase contrast microscope .HASM cell migration was analyzed by using Boyden chamber assay as described previouslyLentiviral transduction of STAT3-short hairpin (sh) RNA (clone ID: V2LHS_262105) in HASM cells was performed as described earlierImmunofluorescence and confocal laser scanning microscopy was performed as we described previouslyLevels of small GTPase Rac1 were measured by a luminescence based G-LISA Rac1 activation assay biochem kit . Briefly, 48 h serum-deprived HASM cells were cultured in presence of TSLP (10 ng/ml) and PDGF (10 ng/ml) and cell lysates were prepared at the indicated times by following the G-LISA kit manufacturer protocol. Luminescence in cell lysates was measured and relative luminescence units (RLU) were determined from experiments performed at least in duplicate.All the data were obtained from experiments performed at least in triplicate. Statistical analysis was performed by using GraphPad Prism Software Version 3.02 for Windows . Data between groups was compared by using one-way analysis of variance (ANOVA) followed by Newman-Keuls multiple comparison test. P values < 0.05 were considered statistically significant.N.S.R. wrote the main manuscript text, N.S.R. and L.S. performed the data analysis, L.S. and A.S.G. designed the experiments, L.S. and H.M. performed the experiments, and A.S.G. provided the reagents, conceived the study design, and critically reviewed, provided feedback and approved the final version of manuscript.


Mice received one topical dose of DMBA, then after one month, two weekly doses of TPA for 14 weeks until sacrifice. RES or UA were applied 20 min prior to DMBA or TPA treatment and 2% dietary CG was given from 2 weeks prior to 2 weeks after the DMBA dose or continually beginning 2 weeks prior to the first dose of TPA. UA applied alone and in combination with CG during the promotion stage was the only inhibitor of tumor multiplicity and tumor incidence. A number of combinations reduced epidermal proliferation, but only UA and the combination UA+CG applied during promotion significantly reduced epidermal hyperplasia. DMBA/TPA application resulted in significant increases in c-jun and p50, which were reversed by a number of different treatments. DMBA/TPA treatment also strongly increased mRNA levels of inflammation markers COX-2 and IL-6. All anti-promotion treatments caused a marked decrease in COX-2 and IL-6 expression compared to the DMBA/TPA control. These results show that UA is a potent inhibitor of skin tumor promotion and inflammatory signaling and it may be useful in the prevention of skin cancer and other epithelial cancers in humans.The purpose of our study was to determine the effect of the combined action of phytochemicals on the early stages of skin tumorigenesis, i.e. initiation and promotion. We tested calcium D-glucarate (CG) given in the diet, while resveratrol (RES) and ursolic acid (UA) were applied topically. The 7,12-dimethylbenz[ The induction of cancer is a multistage process and its stages have been defined experimentally as initiation, promotion, and progression. Carcinogenesis depends on inherited and acquired susceptibility factors, such as mutations in oncogenes and tumor suppressor genes ,2, on exa]anthracene (DMBA) to the skin surface results in Ras mutations in long-lived keratinocytes, probably stem cells (O-tetradecanoylphorbol-13-acetate (TPA), oncogenic transcription factors NFκB and AP-1 are activated  is a naturally occurring phytoalexin associated with many health benefits, most notably the mitigation of age-related diseases, including neurodegeneration, carcinogenesis and atherosclerosis –20. RES ogenesis , as wellogenesis ,26,27. Uogenesis  and breaogenesis  tumorigeogenesis –32.The overall goal of the present study was to determine the effect of combined action of phytochemicals on early stages of skin tumorigenesis, i.e. initiation and promotion. Our hypothesis was that concurrent topical and dietary treatment with selected compounds would lead to more efficient synergistic prevention of chemically-induced murine skin tumorigenesis. Tumors per mouse, tumor incidence, epidermal thickness, epidermal proliferation, and a number of inflammatory biomarkers were measured to determine the effects of these combinations of phytochemicals on the initiation and promotion stages of tumorigenesis.2 skin samples were removed for histology and RNA extraction.Female SENCAR mice, 5 weeks old, were purchased from the National Cancer Institute, Frederick Cancer Research and Development Center . At 6–7 weeks of age the backs of mice were shaved and 20 nmol of DMBA in 0.2 ml acetone (ACT) was applied topically, then after one month, two weekly doses of 2 μg TPA in 0.2 ml of ACT were applied for up to 14 weeks until sacrifice. The phytochemicals RES (2.5 μmol in 0.2 ml ACT) or UA (1 μmol in 0.2 ml DMSO) were applied topically to the dorsal surface of mice 20 min prior to DMBA or TPA treatment and 2% dietary CG was given in the AIN-93G diet from 2 weeks prior until 2 weeks after the DMBA dose or continually beginning 2 weeks prior to the first dose of TPA. The compounds were used in these same concentrations for the combination studies. After the 14th week of treatment, mice were euthanized, tumors were removed and two 1-cmUpon the appearance of papillomas (7th week of TPA treatment) tumors on the backs of each mouse were counted weekly. Tumor multiplicity and tumor incidence were calculated for each group.The tissues were prepared for histological evaluation by using conventional paraffin sections and hematoxylin-eosin staining. The average epithelial thickness was determined from at least 12 randomly selected sites in formalin-fixed skin samples, with 3 measurements per image.For proliferative analysis, mice were given an i.p. injection of BrdU   60 min prior to sacrifice. The tissue sections were immunostained with anti-BrdU antibody . The percentage of stained cells in the basal layer of the epidermis of 12 randomly selected sites was determined. For analysis of NFκB and AP-1 activities, slides were stained with anti-p50 (Lab Vision Co.) or anti-c-jun antibodies . Positive cells were counted as with BrdU samples, however, 3 mice per group were used in the analyses.Ct(Candidate)/2Ct(Control) was used to calculate the normalized ratios.Total RNA was extracted using TRI reagent . RNA (1 mg) was reverse transcribed with oligo(dT), using cMaster RT kit  according to the manufacturer’s protocol. Primers used were: 5′-ATCCTGC CAGCTCCACCG-3′, 5′-TGGTCAAATCCTGTGCTCATA CAT-3′ for COX-2, 5′-GATGTGGAGCAACTTGGAAT-3′, 5′-AGCTCTCCACTTGCAGAAAA-3′ for IL-6 and 5′-CAT CCTGGCCTCGCTGTC-3′, 5′-CTCGTCGTACTCCTGC TTGGT-3′ for β-actin. Standard quantitative RT-PCR was performed in triplicate using SYBR Green RealMasterMix (Eppendorf North America) on the Realplex MasterCycler (Eppendorf). RT-PCR cycle thresholds (Ct) of candidate genes were normalized to control gene β-actin. The formula 2The results for Tumor multiplicity (average papillomas per mouse) and tumor incidence (% of mice with at least one papilloma) for each treatment group were determined by counting papillomas on the backs of mice once per week for the 7th through the 14th week of TPA treatment. No tested compound or combination inhibited carcinogenesis when applied during the initiation (DMBA) phase. In fact, UA significantly increased DMBA/TPA-mediated tumor formation when applied with DMBA in weeks 7 and 9–14 of TPA treatment (p<0.05). However, when applied with TPA, UA inhibited tumor multiplicity by 90% by itself and by 78.9% in CG-fed mice, and both treatments inhibited tumor incidence by 75% at the 14th week of TPA treatment. UA and UA+CG significantly p<0.05 inhibited tumor multiplicity when applied with TPA in each of weeks 7–14 of TPA treatment .The level of epidermal proliferation for each treatment group was determined by counting BrdU-positive cells at 12 random locations per sample, then expressing the data as a percentage of BrdU-positive cells in the basal layer. The proliferation rate of keratinocytes located in the basal layer of epidermis and overall epidermal thickness were greatly increased in the DMBA/TPA treated mice compared to negative control. Despite not having an effect on tumor formation when applied with DMBA, UA alone and in combination with CG was able to inhibit proliferation when applied during the initiation phase, while no compound significantly reduced epidermal hyperplasia when applied with DMBA . All treDMBA/TPA treatments resulted in increased expression of the inflammation markers COX-2 and IL-6, compared to control. These genes play important roles in such inflammatory responses as edema formation and hyperplasia, as well as in papilloma development in mouse skin ,34. WhilAP-1 activation levels were measured by immunostaining for c-jun-positive cells within the interfollicular epidermis. An average of 200 cells per tissue section were counted at 12 random locations per sample, with the data expressed as the mean ± SD of three animals per treatment group. As expected, DMBA/TPA treatment strongly increased the number of c-jun-positive cells. All treatments resulted in a slight decrease of c-jun-positive cells when applied with DMBA . All treNFκB expression for each treatment group was determined by counting p50 protein-positive cells at 12 random locations per sample, then expressing the data as a percentage of positive cells within the interfollicular epidermis. The DMBA/TPA-treated skin presented strong nuclear staining. UA+CG applied during the DMBA initiation stage was the only treatment to significantly suppress the percentage of p50-positive cells . UA alonDMBA/TPA treatments resulted in epidermal hyperplasia, characterized by a significant increase in cells positive for AP-1 and NFκB components with a simultaneously increased expression of the inflammation markers COX-2 and IL-6, compared to untreated controls. Despite our initial hypothesis, we observed no definitive synergistic effect between any of the tested phytochemicals.UA applied alone and in combination with CG during the promotion stage was a strong inhibitor of tumor multiplicity and tumor incidence. While different treatments reduced epidermal proliferation when applied with either DMBA or TPA, only UA and the combination UA+CG applied during promotion significantly reduced hyperplasia. All anti-promotion treatments (either single compounds or their combinations), a majority of single treatments and all combinations during anti-initiation stage caused a marked decrease of inflammation related genes levels compared to the DMBA/TPA control. On the other hand, only UA+CG significantly diminished both c-jun-positive cells and p50-positive cells. These results show that UA alone or in combination with CG is a potent inhibitor of skin tumor promotion and it may be useful in the prevention of skin cancer and other epithelial cancers in humans. In addition, UA+CG is a strong inhibitor of AP-1 and NFκB, and may be useful for the prevention or treatment of maladies associated with activation of these factors.in vivo studies have demonstrated that UA more strongly inhibits TPA-induced phosphorylation and activation of NFκB subunit p65 in vivo (unpublished data). As AP-1 (In our study, we observed UA to be a much stronger inhibitor of tumor formation than RES or CG. This may be because UA and UA+CG were the most potent inhibitors of DMBA/TPA-induced c-jun positive cells and p50-positive cells of the treatments tested. This agrees with studies in other systems, which also show UA is a more potent inhibitor of inflammation-mediated processes such as monocyte recruitment than RES . Our oth As AP-1 ,37 and N As AP-1  activitiNone of the tested treatment groups significantly decreased tumor formation when applied during the DMBA stage. These compounds may only affect traits associated with skin tumor promotion, such as proliferation/clonal expansion, resistance to apoptosis and inflammation. In fact, UA added during the DMBA stage increased tumor formation. This may have been due to UA increasing the reactive concentration of DMBA. UA shows relatively little inhibition of CYP1A1 and CYP1B1, which metabolize polycyclic aromatic hydrocarbons like DMBA to their more reactive form . HoweverOther studies have shown a potent antitumor effect of RES –46, inclThese results indicate UA, alone or in combination with CG, functions as a potent preventer of chemically-induced skin cancer formation. Our previous experiments also identified combinations of natural compounds which synergistically inhibited DMBA-mediated mutation frequency as well as inflammation in the epidermis . Future