Age at Menopause and the Risk of Stroke: Observational and Mendelian Randomization Analysis in 204 244 Postmenopausal Women.

Background Observational studies have shown that women with an early menopause are at higher risk of stroke compared with women with a later menopause. However, associations with stroke subtypes are inconsistent, and the causality is unclear. Methods and Results We analyzed data of the UK Biobank and EPIC-CVD (European Prospective Investigation Into Cancer and Nutrition-Cardiovascular Diseases) study. A total of 204 244 postmenopausal women without a history of stroke at baseline were included (7883 from EPIC-CVD [5292 from the subcohort], 196 361 from the UK Biobank). Pooled mean baseline age was 58.9 years (SD, 5.8), and pooled mean age at menopause was 47.8 years (SD, 6.2). Over a median follow-up of 12.6 years (interquartile range, 11.8-13.3), 6770 women experienced a stroke (5155 ischemic strokes, 1615 hemorrhagic strokes, 976 intracerebral hemorrhages, and 639 subarachnoid hemorrhages). In multivariable adjusted observational Cox regression analyses, the pooled hazard ratios per 5 years younger age at menopause were 1.09 (95% CI, 1.07-1.12) for stroke, 1.09 (95% CI, 1.06-1.13) for ischemic stroke, 1.10 (95% CI, 1.04-1.16) for hemorrhagic stroke, 1.14 (95% CI, 1.08-1.20) for intracerebral hemorrhage, and 1.00 (95% CI, 0.84-1.20) for subarachnoid hemorrhage. When using 2-sample Mendelian randomization analysis, we found no statistically significant association between genetically proxied age at menopause and risk of any type of stroke. Conclusions In our study, earlier age at menopause was related to a higher risk of stroke. We found no statistically significant association between genetically proxied age at menopause and risk of stroke, suggesting no causal relationship.

Diabetes, prediabetes, and atrial fibrillation-A population-based cohort study based on national and regional registers.

BACKGROUND: Previous studies have shown an increased risk for atrial fibrillation and atrial flutter (AF) in people with type 2 diabetes and prediabetes. It is unclear whether this increase in AF risk is independent of other risk factors for AF. OBJECTIVE: To investigate the association between diabetes and different prediabetic states, as independent risk factors for the onset of AF. METHODS: We performed a population-based cohort study in Northern Sweden, including data on fasting plasma glucose, oral glucose tolerance test, major cardiovascular risk factors, medical history, and lifestyle factors. Participants were divided into six groups depending on glycemic status and followed through national registers for AF diagnosis. Cox proportional hazard model was used to assess the association between glycemic status and AF, using normoglycemia as reference. RESULTS: The cohort consisted of 88,889 participants who underwent a total of 139,661 health examinations. In the model adjusted for age and sex, there was a significant association between glycemic status and development of AF in all groups except the impaired glucose tolerance group, with the strongest association for the group with known diabetes (p-value <0.001). In a model adjusted for sex, age, systolic blood pressure, body mass index, antihypertensive drugs, cholesterol, alcohol, smoking, education level, marital status, and physical activity, there was no significant association between glycemic status and AF. CONCLUSIONS/INTERPRETATION: The association between glycemic status and AF disappears upon adjustment for potential confounders. Diabetes and prediabetes do not appear to be independent risk factors for AF.

Macrophage fumarate hydratase restrains mtRNA-mediated interferon production.

Metabolic rewiring underlies the effector functions of macrophages1-3, but the mechanisms involved remain incompletely defined. Here, using unbiased metabolomics and stable isotope-assisted tracing, we show that an inflammatory aspartate-argininosuccinate shunt is induced following lipopolysaccharide stimulation. The shunt, supported by increased argininosuccinate synthase (ASS1) expression, also leads to increased cytosolic fumarate levels and fumarate-mediated protein succination. Pharmacological inhibition and genetic ablation of the tricarboxylic acid cycle enzyme fumarate hydratase (FH) further increases intracellular fumarate levels. Mitochondrial respiration is also suppressed and mitochondrial membrane potential increased. RNA sequencing and proteomics analyses demonstrate that there are strong inflammatory effects resulting from FH inhibition. Notably, acute FH inhibition suppresses interleukin-10 expression, which leads to increased tumour necrosis factor secretion, an effect recapitulated by fumarate esters. Moreover, FH inhibition, but not fumarate esters, increases interferon-ß production through mechanisms that are driven by mitochondrial RNA (mtRNA) release and activation of the RNA sensors TLR7, RIG-I and MDA5. This effect is recapitulated endogenously when FH is suppressed following prolonged lipopolysaccharide stimulation. Furthermore, cells from patients with systemic lupus erythematosus also exhibit FH suppression, which indicates a potential pathogenic role for this process in human disease. We therefore identify a protective role for FH in maintaining appropriate macrophage cytokine and interferon responses.

Type I interferon receptor signalling deficiency results in dysregulated innate immune responses to SARS-CoV-2 in mice.

SARS-CoV-2 is a newly emerged coronavirus, causing the global pandemic of respiratory coronavirus disease (COVID-19). The type I interferon (IFN) pathway is of particular importance for anti-viral defense and recent studies identified that type I IFNs drive early inflammatory responses to SARS-CoV-2. Here, we use a mouse model of SARS-CoV-2 infection, facilitating viral entry by intranasal recombinant Adeno-Associated Virus (rAAV) transduction of hACE2 in wildtype (WT) and type I IFN receptor-1 deficient (Ifnar1-/- ) mice, to study the role of type I IFN signalling and innate immune responses during SARS-CoV-2 infection. Our data show that type I IFN signalling is essential for inducing anti-viral effector responses to SARS-CoV-2, control of virus replication, and to prevent enhanced disease. Furthermore, hACE2-Ifnar1-/- mice had increased gene expression of the chemokine Cxcl1 and airway infiltration of neutrophils as well as reduced and delayed production of monocyte-recruiting chemokine CCL2. hACE2-Ifnar1-/- mice showed altered recruitment of inflammatory myeloid cells to the lung upon SARS-CoV-2 infection, with a shift from Ly6C+ to Ly6C- expressing cells. Together, our findings suggest that type I IFN signalling deficiency results in a dysregulated innate immune response to SARS-CoV-2 infection.

Genomic and Phenotypic Characteristics in Geographically Separated Clinical Campylobacter jejuni ST353CC Isolates.

Campylobacter jejuni fecal isolates of eight international travelers, 5 of which had traveled to Ecuador and 3 to Bangladesh, were characterized, and the possible relationship between bacterial traits and clinical symptoms was further analyzed. All eight isolates belonged to the same Multi-Locus Sequence Type clonal complex (ST353CC). The three isolates from Bangladesh were all of the same sequence type (ST-9438), and when compared to isolates of various other sequence types, they had a larger quantity of unique genetic content, higher expression levels of some putative virulence genes involved in adhesion and invasion (flpA, ciaB and iamA), and showed higher adhesion levels to human HT-29 colon cancer cells in an in vitro infection model. However, in contrast to the seemingly higher pathogenic potential of these bacterial isolates, travelers infected with the ST-9438 isolates had no or only very mild symptoms, whereas the other individuals, whose bacterial isolates seemed to have less pathogenic potential, generally reported severe symptoms. When studying the 16S rRNA gene-based fecal microbiota in samples collected prior to travel, there was an individual variation in the relative abundance of the three major bacterial phyla Actinobacteria, Bacteroidetes and Firmicutes, but there were no associations between composition and diversity of microbiota and development of severe symptoms from the infection. It remains to be confirmed by larger studies whether an individual's characteristics such as gut microbiota, might be related to the severity of symptoms in Campylobacter infections.

Lentiviral and AAV-mediated expression of palivizumab offer protection against Respiratory Syncytial Virus infection.

Respiratory syncytial virus (RSV) infection is a common cause of hospitalisation in infants and the elderly. Palivizumab prophylaxis is the only approved treatment modality but is costly and only offered to select vulnerable populations. Here, we investigated gene delivery approaches via recombinant adeno-associated virus (rAAV2/8) and simian immunodeficiency virus (rSIV.F/HN) vectors to achieve sustained in vivo production of palivizumab in a murine model. Delivery of palivizumab-expressing vectors 28 days prior to RSV challenge resulted in complete protection from RSV-induced weight loss. This approach offers prophylaxis against RSV infection, allowing for wider use and reduction in treatment costs in vulnerable populations.

R848 or influenza virus can induce potent innate immune responses in the lungs of neonatal mice.

Innate immune responses are important to protect the neonatal lung, which becomes exposed to commensal and pathogenic microorganisms immediately after birth, at a time when both the lung and the adaptive immune system are still developing. How immune cells in the neonatal lung respond to innate immune stimuli, including toll-like receptor (TLR) agonists, or viruses, is currently unclear. To address this, adult and neonatal mice were intranasally administered with various innate immune stimuli, respiratory syncytial virus (RSV) or influenza virus and cytokine and chemokine levels were quantified. The neonatal lungs responded weakly to RSV and most stimuli but more strongly than adult mice to R848 and influenza virus, both of which activate TLR7 and the inflammasome. Notably, neonatal lungs also contained higher levels of cAMP, a secondary messenger produced following adenosine receptor signaling, than adult lungs and increased responsiveness to R848 was observed in adult mice when adenosine was coadministered. Our data suggest that the neonatal lung may respond preferentially to stimuli that coactivate TLR7 and the inflammasome and that these responses may be amplified by extracellular adenosine. Improved understanding of regulation of immune responses in the neonatal lung can inform the development of vaccine adjuvants for the young.

Neutrophilic inflammation in the respiratory mucosa predisposes to RSV infection.

The variable outcome of viral exposure is only partially explained by known factors. We administered respiratory syncytial virus (RSV) to 58 volunteers, of whom 57% became infected. Mucosal neutrophil activation before exposure was highly predictive of symptomatic RSV disease. This was associated with a rapid, presymptomatic decline in mucosal interleukin-17A (IL-17A) and other mediators. Conversely, those who resisted infection showed presymptomatic activation of IL-17- and tumor necrosis factor-related pathways. Vulnerability to infection was not associated with baseline microbiome but was reproduced in mice by preinfection chemokine-driven airway recruitment of neutrophils, which caused enhanced disease mediated by pulmonary CD8+ T cell infiltration. Thus, mucosal neutrophilic inflammation at the time of RSV exposure enhances susceptibility, revealing dynamic, time-dependent local immune responses before symptom onset and explaining the as-yet unpredictable outcomes of pathogen exposure.

Neutrophils do not impact viral load or the peak of disease severity during RSV infection.

Lung and airway neutrophils are a hallmark of severe disease in infants with respiratory syncytial virus (RSV)-induced lower respiratory tract infections. Despite their abundance in the lungs during RSV infection of both mice and man, the role of neutrophils in viral control and in immune pathology is not clear. Here, antibody mediated neutrophil depletion was used to investigate the degree to which neutrophils impact the lung immune environment, the control of viral replication and the peak severity of disease after RSV infection of mice. Neutrophil depletion did not substantially affect the levels of inflammatory mediators such as type I interferons, IL-6, TNF-α or IL-1ß in response to RSV. In addition, the lack of neutrophils did not change the viral load during RSV infection. Neither neutrophil depletion nor the enhancement of lung neutrophils by administration of the chemoattractant CXCL1 during RSV infection affected disease severity as measured by weight loss. Therefore, in this model of RSV infection, lung neutrophils do not offer obvious benefits to the host in terms of increasing anti-viral inflammatory responses or restricting viral replication and neutrophils do not contribute to disease severity.

Differences in virulence gene expression between human blood and stool Campylobacter coli clade 1 ST828CC isolates.

BACKGROUND: Campylobacter colonise the gastrointestinal tract of warm-blooded animals and are major enteropathogens in humans. C. coli is less common than C. jejuni and accounts for about 10% of the total number of Campylobacter infections although the two species seem to share many virulence determinants. Campylobacter bacteraemia is rare, estimated to occur in less than 1% of the infections, and the exact mechanisms regulating the progression of the infection from the gastrointestinal tract to the blood stream are unclear. Here, we looked at the contribution of C. coli to Campylobacter infections and further compared various virulence traits in C. coli clade 1 blood and stool isolates. RESULTS: We assessed the numbers of C. jejuni and C. coli among typed isolates in the PubMLST database and found that C. coli accounted for 25.9% of blood isolates, but only 8.9% of the stool isolates. Phylogenetic analysis of 128 C. coli clade 1 whole genome sequences deposited to NCBI revealed no specific clustering of the human blood, stool or animal isolates. Of the six C. coli isolates chosen for phenotypic analyses, stool isolates adhered significantly better to human HT-29 colon cancer cells than the blood isolates, while there was no difference in induced IL-8 levels between the isolates. Furthermore, the stool isolates had two- to fourfold higher RNA expression levels of the flpA, ciaB, iamA and cdt virulence genes than the blood isolates. Finally, we looked at the gene structure of the cdtA, B and C toxin genes and found numerous nucleotide additions and deletions disrupting the open reading frames. In contrast to 58% isolates of animal origin, only 38% and 32% of human blood and stool isolates, respectively, had all three cdt genes intact, a prerequisite to produce functional toxins. CONCLUSIONS: This study reveals interesting differences between C. coli clade 1 isolates of human and animal origin on one hand, and also between human blood and stool isolates, on the other. The results suggest that C. coli might downregulate and/or inactivate various virulence determinants as the isolates pass from the animal host to the human gastrointestinal tract and enter the human blood stream.

Campylobacter coli Clade 3 Isolates Induce Rapid Cell Death In Vitro.

Campylobacter bacteria are major human enteropathogens. Campylobacter coli shows less genetic diversity than C. jejuni and clusters into three clades, of which clade 1 includes most human and farm animal isolates, while environmental C. coli isolates mainly belong to clades 2 and 3. Recently, we sequenced the whole genomes of eight C. coli clade 2 and 3 isolates cultivated from water, and here we studied their interaction with human HT-29 colon cancer cells compared to that of clinical clade 1 isolates. All C. coli clade 3 isolates already caused cell necrosis 1 to 2 h after inoculation, whereas none of the clade 1 and 2 isolates analyzed induced cell death. Isolates from clades 2 and 3 adhered to epithelial cells better than clade 1 isolates, but all isolates induced similar levels of interleukin-8 (IL-8). Alignment and phylogenetic analysis of the translated putative virulence genes cadF, flpA, iamA, ciaB, and ceuE revealed clade-specific protein sequence variations, with clade 1 and 2 sequences being more closely related and clade 3 sequences being further apart, in general. Moreover, when RNA levels were measured, clade 3 isolates showed significantly lower levels of expression of cadF, iamA, and ceuE than clade 2 isolates, while flpA expression levels were higher in clade 3 isolates. The cytolethal distending toxin genes were also expressed in clades 2 and 3, although there was no difference between clades. Our findings demonstrate differences between the effects of C. coli clade 1, 2, and 3 isolates on human cells and suggest that C. coli clade 3 might be more virulent than clade 2 due to the observed cytotoxicity.IMPORTANCECampylobacter coli is a common zoonotic cause of gastroenteritis in humans worldwide. The majority of infections are caused by C. coli clade 1 isolates, whereas infections due to clade 2 and 3 isolates are rare. Whether this depends on a low prevalence of clade 2 and 3 isolates in reservoirs important for human infections or their lower ability to cause human disease is unknown. Here, we studied the effects of C. coli clade 2 and 3 isolates on a human cell line. These isolates adhered to human cells to a higher degree than clinical clade 1 isolates. Furthermore, we could show that C. coli clade 3 isolates rapidly induced cell death, suggesting differences in the virulence of C. coli The exact mechanism of cell death remains to be revealed, but selected genes showed interesting clade-specific expression patterns.

Genomic and phenotypic characteristics of Swedish C. jejuni water isolates.

Campylobacter jejuni is the most common cause of bacterial gastroenteritis. Major reservoirs are warm-blooded animals, poultry in particular, but Campylobacter can also be transmitted via water. In this paper, we have taken a closer look at the biology and potential virulence of C. jejuni water isolates. Seven C. jejuni isolates from incoming surface water at water plants in Sweden were characterized with whole genome sequencing and phenotypical testing. Multi locus sequence typing analysis revealed that these isolates belonged to groups known to include both common (ST48CC) and uncommon (ST1275CC, ST683, ST793 and ST8853) human pathogens. Further genomic characterization revealed that these isolates had potential for arsenic resistance (due to presence of arsB gene in all isolates), an anaerobic dimethyl sulfoxide oxidoreductase (in three isolates) and lacked the MarR-type transcriptional regulator gene rrpB (in all but one isolate) earlier shown to be involved in better survival under oxidative and aerobic stress. As putative virulence factors were concerned, there were differences between the water isolates in the presence of genes coding for cytolethal distending toxin (cdtABC), Type VI secretion system and sialylated LOS, as well as in biofilm formation. However, all isolates were motile and could adhere to and invade the human HT-29 colon cancer cell line in vitro and induce IL-8 secretion suggesting potential to infect humans. This is, to the best of our knowledge, the first study where C. jejuni water isolates have been characterized using whole genome sequencing and phenotypical assays. We found differences and shared traits among the isolates but also potential to infect humans.

Heterogeneous Nuclear Ribonucleoprotein C Proteins Interact with the Human Papillomavirus Type 16 (HPV16) Early 3'-Untranslated Region and Alleviate Suppression of HPV16 Late L1 mRNA Splicing.

In order to identify cellular factors that regulate human papillomavirus type 16 (HPV16) gene expression, cervical cancer cells permissive for HPV16 late gene expression were identified and characterized. These cells either contained a novel spliced variant of the L1 mRNAs that bypassed the suppressed HPV16 late, 5'-splice site SD3632; produced elevated levels of RNA-binding proteins SRSF1 (ASF/SF2), SRSF9 (SRp30c), and HuR that are known to regulate HPV16 late gene expression; or were shown by a gene expression array analysis to overexpress the RALYL RNA-binding protein of the heterogeneous nuclear ribonucleoprotein C (hnRNP C) family. Overexpression of RALYL or hnRNP C1 induced HPV16 late gene expression from HPV16 subgenomic plasmids and from episomal forms of the full-length HPV16 genome. This induction was dependent on the HPV16 early untranslated region. Binding of hnRNP C1 to the HPV16 early, untranslated region activated HPV16 late 5'-splice site SD3632 and resulted in production of HPV16 L1 mRNAs. Our results suggested that hnRNP C1 controls HPV16 late gene expression.

Acetylation of intragenic histones on HPV16 correlates with enhanced HPV16 gene expression.

We report that many histone modifications are unevenly distributed over the HPV16 genome in cervical cancer cells as well as in HPV16-immortalized keratinocytes. For example, H3K36me3 and H3K9Ac that are common in highly expressed cellular genes and over exons, were more common in the early than in the late region of the HPV16 genome. In contrast, H3K9me3, H4K20me3, H2BK5me1 and H4K16Ac were more frequent in the HPV16 late region. Furthermore, a region encompassing the HPV16 early polyadenylation signal pAE displayed high levels of histone H3 acetylation. Histone deacetylase (HDAC) inhibitors caused a 2- to 8-fold induction of HPV16 early and late mRNAs in cervical cancer cells and in immortalized keratinocytes, while at the same time increasing the levels of acetylated histones in the cells and on the HPV16 genome specifically. We concluded that increased histone acetylation on the HPV16 genome correlates with increased HPV16 gene expression.

DNGR-1 is dispensable for CD8+ T-cell priming during respiratory syncytial virus infection.

During respiratory syncytial virus (RSV) infection CD8(+) T cells both assist in viral clearance and contribute to immunopathology. CD8(+) T cells recognize viral peptides presented by dendritic cells (DCs), which can directly present viral antigens when infected or, alternatively, "cross-present" antigens after endocytosis of dead or dying infected cells. Mouse CD8α(+) and CD103(+) DCs excel at cross-presentation, in part because they express the receptor DNGR-1 that detects dead cells by binding to exposed F-actin and routes internalized cell debris into the cross-presentation pathway. As RSV causes death in infected epithelial cells, we tested whether cross-presentation via DNGR-1 is necessary for CD8(+) T-cell responses to the virus. DNGR-1-deficient or wild-type mice were intranasally inoculated with RSV and the magnitude of RSV-specific CD8(+) T-cell induction was measured. We found that during live RSV infection, cross-presentation via DNGR-1 did not have a major role in the generation of RSV-specific CD8(+) T-cell responses. However, after intranasal immunization with dead cells infected with RSV, a dependence on DNGR-1 for RSV-specific CD8(+) T-cell responses was observed, confirming the ascribed role of the receptor. Thus, direct presentation by DCs may be the major pathway initiating CD8(+) T-cell responses to RSV, while DNGR-1-dependent cross-presentation has no detectable role.

Suppression of HPV-16 late L1 5'-splice site SD3632 by binding of hnRNP D proteins and hnRNP A2/B1 to upstream AUAGUA RNA motifs.

Human papillomavirus type 16 (HPV-16) 5'-splice site SD3632 is used exclusively to produce late L1 mRNAs. We identified a 34-nt splicing inhibitory element located immediately upstream of HPV-16 late 5'-splice site SD3632. Two AUAGUA motifs located in these 34 nt inhibited SD3632. Two nucleotide substitutions in each of the HPV-16 specific AUAGUA motifs alleviated splicing inhibition and induced late L1 mRNA production from episomal forms of the HPV-16 genome in primary human keratinocytes. The AUAGUA motifs bind specifically not only to the heterogeneous nuclear RNP (hnRNP) D family of RNA-binding proteins including hnRNP D/AUF, hnRNP DL and hnRNP AB but also to hnRNP A2/B1. Knock-down of these proteins induced HPV-16 late L1 mRNA expression, and overexpression of hnRNP A2/B1, hnRNP AB, hnRNP DL and the two hnRNP D isoforms hnRNP D37 and hnRNP D40 further suppressed L1 mRNA expression. This inhibition may allow HPV-16 to hide from the immune system and establish long-term persistent infections with enhanced risk at progressing to cancer. There is an inverse correlation between expression of hnRNP D proteins and hnRNP A2/B1 and HPV-16 L1 production in the cervical epithelium, as well as in cervical cancer, supporting the conclusion that hnRNP D proteins and A2/B1 inhibit HPV-16 L1 mRNA production.

Eight nucleotide substitutions inhibit splicing to HPV-16 3'-splice site SA3358 and reduce the efficiency by which HPV-16 increases the life span of primary human keratinocytes.

The most commonly used 3'-splice site on the human papillomavirus type 16 (HPV-16) genome named SA3358 is used to produce HPV-16 early mRNAs encoding E4, E5, E6 and E7, and late mRNAs encoding L1 and L2. We have previously shown that SA3358 is suboptimal and is totally dependent on a downstream splicing enhancer containingmultiple potential ASF/SF2 binding sites. Here weshow that only one of the predicted ASF/SF2 sites accounts for the majority of the enhancer activity. We demonstrate that single nucleotide substitutions in this predicted ASF/SF2 site impair enhancer function and that this correlates with less efficient binding to ASF/SF2 in vitro. We provide evidence that HPV-16 mRNAs that arespliced to SA3358 interact with ASF/SF2 in living cells. In addition,mutational inactivation of the ASF/SF2 site weakened the enhancer at SA3358 in episomal forms of the HPV-16 genome, indicating that the enhancer is active in the context of the full HPV-16 genome.This resulted in induction of HPV-16 late gene expression as a result of competition from late splice site SA5639. Furthermore, inactivation of the ASF/SF2 site of the SA3358 splicing enhancer reduced the ability of E6- and E7-encoding HPV-16 plasmids to increase the life span of primary keratinocytes in vitro, demonstrating arequirement for an intact splicing enhancer of SA3358 forefficient production of the E6 and E7 mRNAs. These results link the strength of the HPV-16 SA3358 splicing enhancer to expression of E6 and E7 and to the pathogenic properties of HPV-16.

Regulation of human papillomavirus gene expression by splicing and polyadenylation.

Human papillomaviruses (HPVs) are small DNA tumour viruses that are present in more than 99% of all cervical cancers. The ability of these viruses to cause disease is partly attributed to the strict coordination of viral gene expression with the differentiation stage of the infected cell. HPV gene expression is regulated temporally at the level of RNA splicing and polyadenylation, and a dysregulated gene expression programme allows some HPV types to establish long-term persistence, which is a risk factor for cancer. In this Review, we summarize the role of splicing and polyadenylation in the regulation of HPV gene expression and discuss the viral and cellular factors that control these processes.

High expression of arachidonate 15-lipoxygenase and proinflammatory markers in human ischemic heart tissue.

A common feature of the ischemic heart and atherosclerotic plaques is the presence of hypoxia (insufficient levels of oxygen in the tissue). Hypoxia has pronounced effects on almost every aspect of cell physiology, and the nuclear transcription factor hypoxia inducible factor-1α (HIF-1α) regulates adaptive responses to low concentrations of oxygen in mammalian cells. In our recent work, we observed that hypoxia increases the proinflammatory enzyme arachidonate 15-lipoxygenase (ALOX15B) in human carotid plaques. ALOX15 has recently been shown to be present in the human myocardium, but the effect of ischemia on its expression has not been investigated. Here we test the hypothesis that ischemia of the heart leads to increased expression of ALOX15, and found an almost 2-fold increase in HIF-1α mRNA expression and a 17-fold upregulation of ALOX15 mRNA expression in the ischemic heart biopsies from patients undergoing coronary bypass surgery compared with non ischemic heart tissue. To investigate the effect of low oxygen concentration on ALOX15 we incubated human vascular muscle cells in hypoxia and showed that expression of ALOX15 increased 22-fold compared with cells incubated in normoxic conditions. We also observed increased mRNA levels of proinflammatory markers in ischemic heart tissue compared with non-ischemic controls. In summary, we demonstrate increased ALOX15 in human ischemic heart biopsies. Furthermore we demonstrate that hypoxia increases ALOX15 in human muscle cells. Our results yield important insights into the underlying association between hypoxia and inflammation in the human ischemic heart disease.

HPV-16 E2 contributes to induction of HPV-16 late gene expression by inhibiting early polyadenylation.

We provide evidence that the human papillomavirus (HPV) E2 protein regulates HPV late gene expression. High levels of E2 caused a read-through at the early polyadenylation signal pAE into the late region of the HPV genome, thereby inducing expression of L1 and L2 mRNAs. This is a conserved property of E2 of both mucosal and cutaneous HPV types. Induction could be reversed by high levels of HPV-16 E1 protein, or by the polyadenylation factor CPSF30. HPV-16 E2 inhibited polyadenylation in vitro by preventing the assembly of the CPSF complex. Both the N-terminal and hinge domains of E2 were required for induction of HPV late gene expression in transfected cells as well as for inhibition of polyadenylation in vitro. Finally, overexpression of HPV-16 E2 induced late gene expression from a full-length genomic clone of HPV-16. We speculate that the accumulation of high levels of E2 during the viral life cycle, not only turns off the expression of the pro-mitotic viral E6 and E7 genes, but also induces the expression of the late HPV genes L1 and L2.