Deciphering the heterogeneity of the Lyve1+ perivascular macrophages in the mouse brain.

Perivascular macrophages (pvMs) are associated with cerebral vasculature and mediate brain drainage and immune regulation. Here, using reporter mouse models, whole brain and section immunofluorescence, flow cytometry, and single cell RNA sequencing, besides the Lyve1+F4/80+CD206+CX3CR1+ pvMs, we identify a CX3CR1- pvM population that shares phagocytic functions and location. Furthermore, the brain parenchyma vasculature mostly hosts Lyve1+MHCII- pvMs with low to intermediate CD45 expression. Using the double Cx3cr1GFP x Cx3cr1-Cre;RosatdT reporter mice for finer mapping of the lineages, we establish that CD45lowCX3CR1- pvMs are derived from CX3CR1+ precursors and require PU.1 during their ontogeny. In parallel, results from the Cxcr4-CreErt2;Rosa26tdT lineage tracing model support a bone marrow-independent replenishment of all Lyve1+ pvMs in the adult mouse brain. Lastly, flow cytometry and 3D immunofluorescence analysis uncover increased percentage of pvMs following photothrombotic induced stroke. Our results thus show that the parenchymal pvM population is more heterogenous than previously described, and includes a CD45low and CX3CR1- pvM population.

Amiodarone-induced Ataxia: A Case Report of Severe Cerebellar Dysfunction and Review of Literature.

Amiodarone therapy is widely prescribed in patients with atrial fibrillation. The higher prevalence of this arrhythmic heart disease, and the specific age-related issues of homeostasis in the elderly population, makes this group particularly exposed to its adverse effects. Among the many described side-effects, neurological impairments are the less documented and studied. Because amiodarone can be responsible for severe complications, as described in the case below, a close monitoring is necessary throughout its prescription. Awareness should be brought on the amiodarone-induced neurological side-effects as they could be overlooked.

Cerebellar parcellation in schizophrenia and bipolar disorder.

OBJECTIVE: The cerebellum is involved in cognitive processing and emotion control. Cerebellar alterations could explain symptoms of schizophrenia spectrum disorder (SZ) and bipolar disorder (BD). In addition, literature suggests that lithium might influence cerebellar anatomy. Our aim was to study cerebellar anatomy in SZ and BD, and investigate the effect of lithium. METHODS: Participants from 7 centers worldwide underwent a 3T MRI. We included 182 patients with SZ, 144 patients with BD, and 322 controls. We automatically segmented the cerebellum using the CERES pipeline. All outputs were visually inspected. RESULTS: Patients with SZ showed a smaller global cerebellar gray matter volume compared to controls, with most of the changes located to the cognitive part of the cerebellum (Crus II and lobule VIIb). This decrease was present in the subgroup of patients with recent-onset SZ. We did not find any alterations in the cerebellum in patients with BD. However, patients medicated with lithium had a larger size of the anterior cerebellum, compared to patients not treated with lithium. CONCLUSION: Our multicenter study supports a distinct pattern of cerebellar alterations in SZ and BD.

Potential dietary feed additives with antibacterial effects and their impact on performance of weaned piglets: A meta-analysis.

This meta-analysis evaluated the use of potential dietary feed additives (pDFA) with antibacterial effects and their impact on the perfomance of weaned piglets. Twenty-three peer-reviewed in vivo studies, comprising 50 trials, were identified between January 2010 and January 2017. The pDFA in these studies could be grouped in 5 classes: antimicrobial peptides, chitosan, lysozyme, medium chain fatty acids/ triglycerides and plant extracts. Mixed-effect meta-analyses with type of pDFA as fixed effect were performed for the growth parameters 'average daily gain' (ADG) and 'feed conversion ratio' (FCR), which are the two most important and used economic performance parameters for farmers. For each class of pDFA, results of the meta-analysis showed significantly higher average daily gain in the group with pDFA compared to the negative control group, while no significant difference with the positive control group was observed. Furthermore, a positive effect on FCR was found, i.e. significantly less feed was needed to gain 1 kg of body weight in the group with pDFA compared to the negative control group. No significant differences with positive control groups were observed for each class of pDFA, except for plant extracts, where the FCR was also significantly reduced in the treatment group. These results suggest that pDFA could reduce the use of antimicrobials without significant negative effects on performance indicators.

Biosecurity practices in Belgian cattle farming: Level of implementation, constraints and weaknesses.

Over the last few years, a shift from curative towards preventive medicine occurred in the livestock sector. This led to an increased importance of biosecurity to better control infectious diseases by preventing their introduction and/or reducing their spread. Farmers are the main responsible actors of biosecurity measures (BSM). Existing studies report a low implementation level of BSM by the cattle farmers. Barriers such as cost, usefulness, importance, workload and lack of knowledge were investigated but the decision-making process of farmers related to a given BSM is not yet clarified. The objectives of this study were to (i) assess the level of implementation of BSM in cattle farms, (ii) assess the correlation between the importance that farmers give to a BSM and its effective implementation and (iii) identify the main reasons of non-implementation. A randomized survey was implemented in Belgium from December 2016 up to April 2017 with face-to-face interviews conducted in 100 Belgian farms. A descriptive analysis of data was performed using Microsoft Excel® and Stata14® . Chi-square and Spearman's rank correlation tests, respectively, allowed comparing implementation levels in dairy herds vs. beef herds and investigating the correlation between the importance that farmers give to a BSM and its implementation level. Biosecurity measures were poorly implemented to prevent disease introduction through direct contact and almost not to avoid indirect transmission. Some measures showed a significant difference in terms of implementation level between beef and dairy herds. A positive correlation was highlighted between the importance that farmers give to a BSM and its actual effective implementation. Perceived lack of efficiency, feasibility and usefulness are the reasons most often mentioned for non-implementation. Other factors potentially influencing the decision-making process should be further investigated and clarified. Evidence-based studies would be useful to convince the farmers of the need of implementing BSM.

Review of transmission routes of 24 infectious diseases preventable by biosecurity measures and comparison of the implementation of these measures in pig herds in six European countries.

This study aimed to review the transmission routes of important infectious pig diseases and to translate these into biosecurity measures preventing or reducing the transmission between and within pig herds. Furthermore, it aimed to identify the level of implementation of these measures in different European countries and discuss the observed variations to identify potentials for improvement. First, a literature review was performed to show which direct and indirect transmission routes of 24 infectious pig diseases can be prevented through different biosecurity measures. Second, a quantitative analysis was performed using the Biocheck.UGent™, a risk-based scoring system to evaluate biosecurity in pig herds, to obtain an insight into the implementation of these biosecurity measures. The database contained farm-specific biosecurity data from 574 pig farms in Belgium, Denmark, France, Germany, the Netherlands and Sweden, entered between January 2014 and January 2016. Third, a qualitative analysis based on a review of literature and other relevant information resources was performed for every subcategory of internal and external biosecurity in the Biocheck.UGent™ questionnaire. The quantitative analysis indicated that at the level of internal, external and overall biosecurity, Denmark had a significantly distinct profile with higher external biosecurity scores and less variation than the rest of the countries. This is likely due to a widely used specific pathogen-free (SPF) system with extensive focus on biosecurity since 1971 in Denmark. However, the observed pattern may also be attributed to differences in data collection methods. The qualitative analysis identified differences in applied policies, legislation, disease status, pig farm density, farming culture and habits between countries that can be used for shaping country-specific biosecurity advice to attain improved prevention and control of important pig diseases in European pig farms.

Sow and piglet factors determining variation of colostrum intake between and within litters.

Colostrum intake has a short- and long-term beneficial impact on piglet performance and mortality. Sows' colostrum production and piglets' colostrum intake are limited and highly variable. The present study investigated sow and piglet factors explaining the variation of colostrum intake between and within litters. The CV for colostrum intake and birth weight (BWb) of all piglets within a litter was calculated to evaluate the variation of colostrum intake and BWb within a litter (colostrum and litter BWb heterogeneity, respectively). A total of 1937 live-born piglets from 135 litters from 10 commercial herds were included. Colostrum intake per piglet averaged 371±144 g and was affected by breed (P=0.02). It was lower when oxytocin was administered to the sow during parturition (P=0.001) and with increased litter size (P<0.001). It was higher when the interval between birth and first suckling decreased (t FS, P<0.001). Colostrum intake was positively influenced by BWb (P<0.001) and this association was more pronounced in piglets from Topigs (P=0.03) and Hypor (P=0.03) sows compared with piglets from Danbred sow breeds. The positive relationship between colostrum intake and BWb was more pronounced when t FS lasted longer (P=0.009). Heterogeneity in colostrum intake averaged 31±11%, it increased when oxytocin was applied during farrowing (P=0.004) and when stillbirth occurred (P=0.006). Colostrum heterogeneity was positively associated with litter size (P<0.001) and litter BWb heterogeneity (P=0.01). The positive relationship between colostrum and litter BWb heterogeneity was more pronounced when oxytocin was applied during farrowing (P=0.04). The present study demonstrated that oxytocin should be used cautiously in sows during farrowing. Farrowing and colostrum management should prevent or counteract the adverse influences of stillbirth, large and heterogeneous litters on colostrum intake and colostrum heterogeneity. The study also confirmed the expected association between BWb and colostrum intake and indicated that the impact of BWb on colostrum intake was different among breeds (Hypor v. Danbred) and dependent on piglets' latency to first suckling. Hence, colostrum management should focus on low birth weight piglets, especially in some breeds, and low colostrum intake in low birth weight piglets can be counteracted by shortening the t FS.

Reducing Antimicrobial Usage in Pig Production without Jeopardizing Production Parameters.

Antimicrobial usage (AMU) has been described to be high in pig production. Although farmers are aware of the high usage, little is known about intervention to improve the situation. This study evaluated the extent to which AMU could be reduced in pig production by the optimization of herd management, biosecurity status, vaccination strategy, anthelmintic therapy and advice on prudent AMU. Furthermore, the effects of these interventions on the herd production results were explored. This intervention study was conducted on 61 Flemish pig herds and included three visits per herd. During the initial visit, information was gathered on herd management, biosecurity status (quantified by means of the Biocheck.UGent™ risk-based scoring system), vaccination strategy, anthelmintic therapy and AMU. This info was then translated into a herd-specific action plan which was discussed with the farmer and herd veterinarian/other advisors during the second visit. In the final herd visit (±8 months later), comparable data were obtained to evaluate the progress. Overall, a significant improvement of 2.4 points external and 7 points internal biosecurity on the herds was obtained, combined with additional vaccination, anthelmintic therapy and prudent AMU. This was accompanied by a significant reduction in the AMU with a decrease of 52% for the pigs from birth till slaughter and 32% for breeding animals, based on treatment incidences (TIs) and included an important reduction in the use of critically important antimicrobials. More importantly, the increased biosecurity levels and decreased AMU were combined with significantly improved technical results such as the number of weaned piglets per sow per year (+1.1), daily weight gain (+5.9 g/day) and mortality in the finisher period (-0.6%). Guided interventions as a team effort of farmer and herd veterinarian/other advisors have shown to be a promising method in the reduction of AMU in pig production.

Age and anti-Müllerian hormone levels predict the success of in vitro maturation of cat oocytes.

Up to date, in vitro maturation (IVM) rates of oocytes are highly variable between individual cats. This study was carried out to investigate the predictive value of age and anti-Müllerian hormone (AMH) concentration in relation to capacity for IVM of cat oocytes. Ovaries were collected from 33 cats, which were divided into three age groups: (i) 0-3 months (pre-pubertal); (ii) 3-12 months (peripubertal); and (iii) older than 12 months (pubertal). The cumulus-oocyte complexes (COCs) were matured and subsequently stained to check nuclear maturation status, and blood was taken for AMH analysis. Increasing age was significantly associated with decreasing AMH levels, and mean AMH levels differed significantly between all age categories: group 1: mean AMH 18.71 µg/L; group 2: mean AMH 9.27 µg/L; and group 3: mean AMH 4.13 µg/L. Moreover, the probability of maturation was more likely in groups 2 and 3 compared to group 1. Between categories 2 and 3, no significant difference in maturation probability was found (p = .31). Finally, the probability of oocyte maturation decreased significantly with increasing AMH levels. In age group 2, oocytes with a higher AMH level were less likely to mature. In age groups 1 and 3, no significant association between the AMH level and the proportion of maturated COC was found. We can conclude that if a higher probability of nuclear maturation is required, it is preferable to use cats with lower AMH levels and older than 3 months of age to improve cat IVM.

Long-term effects of colostrum intake in piglet mortality and performance.

The present study investigated the long-term effects of colostrum intake on performance and mortality in pigs. A total of 1,455 live-born piglets in 10 commercial herds were followed from birth until 22 wk of age. Pigs were individually weighed at birth, at weaning, at onset (intermediate weight), and during the fattening period (finishing weight). Colostrum intake was calculated by the mechanistic model developed by Theil et al. (see text for citation). One linear mixed model was fitted to model the possible associations between colostrum intake and weight at the weaning, intermediate, and finishing periods. In addition to colostrum intake as the main predictor of interest, other predictor variables were also tested, namely birth weight, birth order, sex, breed, and the interval between birth and first suckling (t). Colostrum intake and birth weight were positively associated with weaning ( < 0.001), intermediate ( < 0.001), and finishing ( < 0.001) weights. Furthermore, higher colostrum intake is more beneficial to weaning ( < 0.001), intermediate ( < 0.001), and finishing ( = 0.02) weights in piglets with low versus high birth weights. Birth order was positively associated with weight at each measurement time ( = 0.01). Sex affected only finishing weight ( < 0.001). Some breeds differed in piglets' weight at onset or during the fattening period. The association between t and weaning weight differed by breed. Three generalized linear mixed models were performed to model the probability of dying during the suckling, the nursery, or the fattening period. Colostrum intake, birth weight, birth order, sex, breed, and t were tested. Preweaning mortality was negatively associated with colostrum intake ( < 0.001) and birth weight ( = 0.004) and positively associated with t ( < 0.001). Mortality during the nursery period was negatively associated with colostrum intake ( < 0.001) and birth weight ( = 0.002). The negative association between colostrum intake and mortality during the suckling ( < 0.001) and the nursery ( = 0.008) periods was more pronounced in small versus heavy piglets. Mortality during fattening was associated with weaning ( = 0.04) and intermediate ( = 0.006) weight. In conclusion, colostrum intake significantly influences piglets' short-term and long-term performance and mortality. As colostrum yield is reported to be independent of litter size, sufficient colostrum intake per piglet is crucial, especially in hyperprolific sows.

Cerebellar volume in schizophrenia and bipolar I disorder with and without psychotic features.

OBJECTIVE: There is growing evidence that cerebellum plays a crucial role in cognition and emotional regulation. Cerebellum is likely to be involved in the physiopathology of both bipolar disorder and schizophrenia. The objective of our study was to compare cerebellar size between patients with bipolar disorder, patients with schizophrenia, and healthy controls in a multicenter sample. In addition, we studied the influence of psychotic features on cerebellar size in patients with bipolar disorder. METHOD: One hundred and fifteen patients with bipolar I disorder, 32 patients with schizophrenia, and 52 healthy controls underwent 3 Tesla MRI. Automated segmentation of cerebellum was performed using FreeSurfer software. Volumes of cerebellar cortex and white matter were extracted. Analyses of covariance were conducted, and age, sex, and intracranial volume were considered as covariates. RESULTS: Bilateral cerebellar cortical volumes were smaller in patients with schizophrenia compared with patients with bipolar I disorder and healthy controls. We found no significant difference of cerebellar volume between bipolar patients with and without psychotic features. No change was evidenced in white matter. CONCLUSION: Our results suggest that reduction in cerebellar cortical volume is specific to schizophrenia. Cerebellar dysfunction in bipolar disorder, if present, appears to be more subtle than a reduction in cerebellar volume.

[Non-medical applications for brain MRI: Ethical considerations]. / Applications non médicales de l'IRM cérébrale : considérations éthiques.

INTRODUCTION: The recent neuroimaging techniques offer the possibility to better understand complex cognitive processes that are involved in mental disorders and thus have become cornerstone tools for research in psychiatry. The performances of functional magnetic resonance imaging are not limited to medical research and are used in non-medical fields. These recent applications represent new challenges for bioethics. OBJECTIVE: In this article we aim at discussing the new ethical issues raised by the applications of the latest neuroimaging technologies to non-medical fields. METHODS: We included a selection of peer-reviewed English medical articles after a search on NCBI Pubmed database and Google scholar from 2000 to 2013. We screened bibliographical tables for supplementary references. Websites of governmental French institutions implicated in ethical questions were also screened for governmental reports. RESULTS: Findings of brain areas supporting emotional responses and regulation have been used for marketing research, also called neuromarketing. The discovery of different brain activation patterns in antisocial disorder has led to changes in forensic psychiatry with the use of imaging techniques with unproven validity. Automated classification algorithms and multivariate statistical analyses of brain images have been applied to brain-reading techniques, aiming at predicting unconscious neural processes in humans. We finally report the current position of the French legislation recently revised and discuss the technical limits of such techniques. DISCUSSION: In the near future, brain imaging could find clinical applications in psychiatry as diagnostic or predictive tools. However, the latest advances in brain imaging are also used in non-scientific fields raising key ethical questions. Involvement of neuroscientists, psychiatrists, physicians but also of citizens in neuroethics discussions is crucial to challenge the risk of unregulated uses of brain imaging.

Virulence comparison and quantification of horizontal bovine viral diarrhoea virus transmission following experimental infection in calves.

Bovine viral diarrhoea virus (BVDV) causes persistent infections by infecting the fetus of susceptible animals during gestation. These persistently infected (PI) animals are important sources of infection. On the contrary, transiently infected (TI) animals are believed to be less important, but transient infections with a severe BVDV-2 strain can spread explosively. To assess the importance of TI cattle in the epidemiology of BVDV, two experimental infections were performed to determine basic reproduction ratios (R0). In each experiment three calves were infected via intranasal inoculation and housed together with seven susceptible animals. Two strains isolated in Belgium were used, a virulent BVDV-1b and a virulent BVDV-2a field isolate, resulting in an R0 of 0.25 (95% CI 0.01; 1.95) and 0.24 (95% CI 0.01; 2.11), respectively. A PI animal was then introduced to the remaining uninfected animals and produced an R of +∞ (95% CI 1.88; +∞). These results support the suggestion that TI animals, compared to PI animals, contribute only a limited amount to BVDV spread. Additionally, the severe clinical symptoms observed in the field with these isolates could not be reproduced during these experiments, suggesting that other factors besides strain virulence influence the clinical manifestations evoked by BVDV.

Molecular characteristics of Human Endogenous Retrovirus type-W in schizophrenia and bipolar disorder.

Epidemiological and genome-wide association studies of severe psychiatric disorders such as schizophrenia (SZ) and bipolar disorder (BD), suggest complex interactions between multiple genetic elements and environmental factors. The involvement of genetic elements such as Human Endogenous Retroviruses type 'W' family (HERV-W) has consistently been associated with SZ. HERV-W envelope gene (env) is activated by environmental factors and encodes a protein displaying inflammation and neurotoxicity. The present study addressed the molecular characteristics of HERV-W env in SZ and BD. Hundred and thirty-six patients, 91 with BD, 45 with SZ and 73 healthy controls (HC) were included. HERV-W env transcription was found to be elevated in BD (P<10-4) and in SZ (P=0.012) as compared with HC, but with higher values in BD than in SZ group (P<0.01). The corresponding DNA copy number was paradoxically lower in the genome of patients with BD (P=0.0016) or SZ (P<0.0003) than in HC. Differences in nucleotide sequence of HERV-W env were found between patients with SZ and BD as compared with HC, as well as between SZ and BD. The molecular characteristics of HERV-W env also differ from what was observed in Multiple Sclerosis (MS) and may represent distinct features of the genome of patients with BD and SZ. The seroprevalence for Toxoplasma gondii yielded low but significant association with HERV-W transcriptional level in a subgroup of BD and SZ, suggesting a potential role in particular patients. A global hypothesis of mechanisms inducing such major psychoses is discussed, placing HERV-W at the crossroads between environmental, genetic and immunological factors. Thus, particular infections would act as activators of HERV-W elements in earliest life, resulting in the production of an HERV-W envelope protein, which then stimulates pro-inflammatory and neurotoxic cascades. This hypothesis needs to be further explored as it may yield major changes in our understanding and treatment of severe psychotic disorders.

Endocan or endothelial cell specific molecule-1 (ESM-1): a potential novel endothelial cell marker and a new target for cancer therapy.

Endocan, previously called endothelial cell specific molecule-1, is a soluble proteoglycan of 50 kDa, constituted of a mature polypeptide of 165 amino acids and a single dermatan sulphate chain covalently linked to the serine residue at position 137. This dermatan sulphate proteoglycan, which is expressed by the vascular endothelium, has been found freely circulating in the bloodstream of healthy subjects. Experimental evidence is accumulating that implicates endocan as a key player in the regulation of major processes such as cell adhesion, in inflammatory disorders and tumor progression. Inflammatory cytokines such as TNF-alpha, and pro-angiogenic growth factors such as VEGF, FGF-2 and HGF/SF, strongly increased the expression, synthesis or the secretion of endocan by human endothelial cells. Endocan is clearly overexpressed in human tumors, with elevated serum levels being observed in late-stage lung cancer patients, as measured by enzyme-linked immunoassay, and with its overexpression in experimental tumors being evident by immunohistochemistry. Recently, the mRNA levels of endocan have also been recognized as being one of the most significant molecular signatures of a bad prognosis in several types of cancer including lung cancer. Overexpression of this dermatan sulphate proteoglycan has also been shown to be directly involved in tumor progression as observed in mouse models of human tumor xenografts. Collectively, these results suggest that endocan could be a biomarker for both inflammatory disorders and tumor progression as well as a validated therapeutic target in cancer. On the basis of the recent successes of immunotherapeutic approaches in cancer, the preclinical data on endocan suggests that an antibody raised against the protein core of endocan could be a promising cancer therapy.

Tissue-specificity of 412 retrotransposon expression in Drosophila simulans and D. melanogaster.

We analyse the expression of the retrotransposon 412 in the soma, testes, and ovaries in populations of Drosophila simulans and D. melanogaster, using RT-PCR and in situ hybridization. We find that expression of 412 is highly variable in the soma, confirming previous findings based on Northern blots. No 412RNA is detected in the ovaries by either in situ hybridization or RT-PCR, in any population of either species. Transcripts are, however, detected in the male germline, which show a very characteristic spatial pattern of 412 expression in primary spermatocytes. There is no relationship between expression of the 412 element in the soma and in the testes in the populations. These findings show that the expression of 412 is independently regulated in the soma and the testes, and this raises the question of the real influence of the somatic transcripts on the organism and on the transposition rate.

Negative and translation termination-dependent positive control of FLI-1 protein synthesis by conserved overlapping 5' upstream open reading frames in Fli-1 mRNA.

The proto-oncogene Fli-1 encodes a transcription factor of the ets family whose overexpression is associated with multiple virally induced leukemias in mouse, inhibits murine and avian erythroid cell differentiation, and induces drastic perturbations of early development in Xenopus. This study demonstrates the surprisingly sophisticated regulation of Fli-1 mRNA translation. We establish that two FLI-1 protein isoforms (of 51 and 48 kDa) detected by Western blotting in vivo are synthesized by alternative translation initiation through the use of two highly conserved in-frame initiation codons, AUG +1 and AUG +100. Furthermore, we show that the synthesis of these two FLI-1 isoforms is regulated by two short overlapping 5' upstream open reading frames (uORF) beginning at two highly conserved upstream initiation codons, AUG -41 and GUG -37, and terminating at two highly conserved stop codons, UGA +35 and UAA +15. The mutational analysis of these two 5' uORF revealed that each of them negatively regulates FLI-1 protein synthesis by precluding cap-dependent scanning to the 48- and 51-kDa AUG codons. Simultaneously, the translation termination of the two 5' uORF appears to enhance 48-kDa protein synthesis, by allowing downstream reinitiation at the 48-kDa AUG codon, and 51-kDa protein synthesis, by allowing scanning ribosomes to pile up and consequently allowing upstream initiation at the 51-kDa AUG codon. To our knowledge, this is the first example of a cellular mRNA displaying overlapping 5' uORF whose translation termination appears to be involved in the positive control of translation initiation at both downstream and upstream initiation codons.

Spi-1/PU.1 is a positive regulator of the Fli-1 gene involved in inhibition of erythroid differentiation in friend erythroleukemic cell lines.

Spi-1/PU.1 and Fli-1 are two members of the ETS family of transcription factors whose expression is deregulated by proviral insertion in most erythroleukemic cell lines induced by the spleen focus-forming virus (SFFV) and Friend murine leukemia virus (F-MuLV) components of the Friend viral complex, respectively. In this study, we present evidence that transcription of the Fli-1 gene is positively regulated by Spi-1/PU.1 in SFFV-transformed cell lines: (i) all SFFV-transformed cell lines expressing Spi-1/PU.1 are characterized by a specific pattern of Fli-1 gene transcripts initiated in the -200 region instead of position -400 as reported for F-MuLV-transformed cell lines; (ii) these Fli-1 transcripts initiated in the -200 region are downregulated in parallel with that of Spi-1/PU.1 during hexamethylenebisacetamide (HMBA) induced differentiation; and (iii) Fli-1 transcription is upregulated in SFFV cells lines following stable transfection of a Spi-1/PU.1 expression vector. Furthermore, we found by transient transfection assays that the -270/-41 region of the Fli-1 gene displays promoter activity which is transactivated by Spi-1/PU.1. This promoter is strictly dependent on the integrity of two highly conserved ETS DNA binding sites that bind the Spi-1/PU.1 protein in vitro. Finally, we show that transfection of constitutive or inducible Fli-1 expression vectors in SFFV-transformed cells inhibits their erythroid differentiation induced by HMBA. Overall, these data indicate that Fli-1 is a target gene of the Spi-1/PU.1 transcription factor in SFFV-transformed cell lines. We further suggest that deregulated synthesis of Fli-1 may trigger a common mechanism contributing to erythroleukemia induced by either SFFV or F-MuLV.

The time-pattern of rises and falls in proliferation fades with senescence of mortal lines and is perpetuated in immortal rat hepatoma Fao cell line.

Immortal cells perpetuate the rises and falls of proliferation that are progressively damped in mortal long-term cultured cells. For immortal rat hepatoma Fao cells, similar waves of proliferation occurred about every 3-4 wk. Under the same conditions, embryonic human fibroblasts and transformed but not immortalized embryonic fibroblasts display similarly recurring proliferation waves that progressively decrease in amplitude until senescence of the lines. In addition, strains of diploid normal human skin fibroblasts cultured under different culture conditions display a similar time-pattern of proliferation. Although the amplitude and baseline of these fluctuations are characteristic for each cell line, a common point was marked slow down in proliferation after every sequence of about 25 population doublings for all cells. Renewed proliferation waves of Fao cells allow about 22-23 additional population doublings each. Normal embryonic fibroblast culture and its transformed counterpart accumulate about 30 and 60 population doublings, respectively, before senescence. Normal fibroblast strains accumulate about 25 population doublings over their entire life spans. This halt in proliferation after every stretch of about 25 population doublings may correspond to a structural or functional stop following attrition of telomeric DNA. This putative stop may be bypassed once in transformed embryonic cells and repetitively in immortal cells. In support of this hypothesis, we observed rapid telomere shortening, in two steps, during divisions of mortal embryonic cells, and maintenance of long telomeres in immortal Fao cells, which may indicate episodic repair of telomeres. Alternatively, such maintenance of long telomeres may reflect survival and successive clonal growth of rare cells with long telomeres. We suggest that the balance between telomere attrition and repair processes regulates the waves of proliferation.