Zika virus NS3 protease induces bone morphogenetic protein-dependent brain calcification in human fetuses.

The most frequent fetal birth defect associated with prenatal Zika virus (ZIKV) infection is brain calcification, which in turn may potentially affect neurological development in infants. Understanding the mechanism could inform the development of potential therapies against prenatal ZIKV brain calcification. In perivascular cells, bone morphogenetic protein (BMP) is an osteogenic factor that undergoes maturation to activate osteogenesis and calcification. Here, we show that ZIKV infection of cultivated primary human brain pericytes triggers BMP2 maturation, leading to osteogenic gene expression and calcification. We observed extensive calcification near ZIKV+ pericytes of fetal human brain specimens and in vertically transmitted ZIKV+ human signal transducer and activator of transcription 2-knockin mouse pup brains. ZIKV infection of primary pericytes stimulated BMP2 maturation, inducing osteogenic gene expression and calcification that were completely blocked by anti-BMP2/4 neutralizing antibody. Not only did ZIKV NS3 expression alone induce BMP2 maturation, osteogenic gene expression and calcification, but purified NS3 protease also effectively cleaved pro-BMP2 in vitro to generate biologically active mature BMP2. These findings highlight ZIKV-induced calcification where the NS3 protease subverts the BMP2-mediated osteogenic signalling pathway to trigger brain calcification.

[Analysis of the concept of the Zika Virus congenital syndrome]. / Análise do conceito síndrome congênita pelo Zika vírus.

The scope of this article is to analyze the concept of the Zika Virus Congenital Syndrome. It is a conceptual analysis, based on Walker and Avant. In order to operationalize the search, a systematic review was conducted. The essence of the concept of the Zika Virus Congenital Syndrome is determined by the following attributes: intracranial calcification, ventriculomegaly, and diminished brain volume. For this syndrome to occur, it is necessary to have the following antecedents: transplacental transmission of a mother infected by the bite of the Aedes SSP mosquito or by sexual contact. Accordingly, this entails a set of signs and symptoms that go beyond fetal or postnatal microcephaly, such as, for example, delayed neuropsychomotor development, auditory and visual abnormalities, craniofacial disproportion, overlapping cranial sutures, prominent occipital bone, excess nuchal skin, epilepsy, irritability, dyskinesia, hypertonia, hypotonia, hemiplegia, hemiparesis, spasticity and hyperreflexia. The concept of the Zika Virus Congenital Syndrome is newly acknowledged. The presence of the set of signs and symptoms by the Zika Virus Congenital Syndrome is determined by intracranial calcification and decreased brain volume, and the baby may present microcephaly at birth or subsequently.

O objetivo deste artigo é analisar o conceito de Síndrome Congênita pelo Zika Vírus. Trata-se de uma análise de conceito, baseado em Walker e Avant. Para operacionalização da busca foi realizada uma revisão sistemática. A essência do conceito Síndrome Congênita pelo Zika Vírus é determinada pelos atributos: calcificação intracraniana, ventriculomegalia e volume cerebral diminuído. Para que essa síndrome aconteça faz-se necessário que ocorram os antecedentes: transmissão via transplacentária de mãe infectada pela picada do mosquito Aedes SSP ou por via sexual. Com isso, resultam um conjunto de sinais e sintomas além da microcefalia fetal ou pós-natal, como por exemplo, atraso no desenvolvimento neuropsicomotor, anormalidades auditivas e visuais, desproporção craniofacial, suturas cranianas sobrepostas, osso occipital proeminente, excesso de pele nucal, epilepsia, irritabilidade, discinesia, hipertonia, hipotonia, hemiplegia, hemiparesia, espasticidade, hiperreflexia. O conceito de Síndrome Congênita pelo Zika Vírus é recém conhecido. O que determinará a presença do conjunto de sinais e sintomas pela infecção congênita do Zika vírus é a calcificação intracraniana e volume cerebral diminuído, podendo apresentar microcefalia já ao nascer ou apenas posteriormente.

Análise do conceito síndrome congênita pelo Zika vírus / Analysis of the concept of the Zika Virus congenital syndrome

Resumo O objetivo deste artigo é analisar o conceito de Síndrome Congênita pelo Zika Vírus. Trata-se de uma análise de conceito, baseado em Walker e Avant. Para operacionalização da busca foi realizada uma revisão sistemática. A essência do conceito Síndrome Congênita pelo Zika Vírus é determinada pelos atributos: calcificação intracraniana, ventriculomegalia e volume cerebral diminuído. Para que essa síndrome aconteça faz-se necessário que ocorram os antecedentes: transmissão via transplacentária de mãe infectada pela picada do mosquito Aedes SSP ou por via sexual. Com isso, resultam um conjunto de sinais e sintomas além da microcefalia fetal ou pós-natal, como por exemplo, atraso no desenvolvimento neuropsicomotor, anormalidades auditivas e visuais, desproporção craniofacial, suturas cranianas sobrepostas, osso occipital proeminente, excesso de pele nucal, epilepsia, irritabilidade, discinesia, hipertonia, hipotonia, hemiplegia, hemiparesia, espasticidade, hiperreflexia. O conceito de Síndrome Congênita pelo Zika Vírus é recém conhecido. O que determinará a presença do conjunto de sinais e sintomas pela infecção congênita do Zika vírus é a calcificação intracraniana e volume cerebral diminuído, podendo apresentar microcefalia já ao nascer ou apenas posteriormente.

Abstract The scope of this article is to analyze the concept of the Zika Virus Congenital Syndrome. It is a conceptual analysis, based on Walker and Avant. In order to operationalize the search, a systematic review was conducted. The essence of the concept of the Zika Virus Congenital Syndrome is determined by the following attributes: intracranial calcification, ventriculomegaly, and diminished brain volume. For this syndrome to occur, it is necessary to have the following antecedents: transplacental transmission of a mother infected by the bite of the Aedes SSP mosquito or by sexual contact. Accordingly, this entails a set of signs and symptoms that go beyond fetal or postnatal microcephaly, such as, for example, delayed neuropsychomotor development, auditory and visual abnormalities, craniofacial disproportion, overlapping cranial sutures, prominent occipital bone, excess nuchal skin, epilepsy, irritability, dyskinesia, hypertonia, hypotonia, hemiplegia, hemiparesis, spasticity and hyperreflexia. The concept of the Zika Virus Congenital Syndrome is newly acknowledged. The presence of the set of signs and symptoms by the Zika Virus Congenital Syndrome is determined by intracranial calcification and decreased brain volume, and the baby may present microcephaly at birth or subsequently.

Updated Imaging Findings in Congenital Zika Syndrome: A Disease Story That is Still Being Written.

In congenital Zika virus syndrome (CZS), the most frequent radiological findings are calcifications in the cortical-white matter junction and malformations of cortical development (pachygyria or polymicrogyria, which occur predominantly in the frontal lobes, or a simplified gyral pattern), ventriculomegaly, enlargement of the cisterna magna and the extra-axial subarachnoid space, corpus callosum abnormalities, and reduced brain volume. This syndrome can also result in a decrease in the brainstem and cerebellum volumes and delayed myelination. Infants with CZS may show venous thrombosis and lenticulostriate vasculopathies. Over a 3-year follow-up period, many infants with CZS showed hydrocephalus, reduction in brain calcifications, and greater reduction in brain thickness.

Follow-up brain imaging of 37 children with congenital Zika syndrome: case series study.

Objective To compare initial brain computed tomography (CT) scans with follow-up CT scans at one year in children with congenital Zika syndrome, focusing on cerebral calcifications.Design Case series study.Setting Barão de Lucena Hospital, Pernambuco state, Brazil.Participants 37 children with probable or confirmed congenital Zika syndrome during the microcephaly outbreak in 2015 who underwent brain CT shortly after birth and at one year follow-up.Main outcome measure Differences in cerebral calcification patterns between initial and follow-up scans.Results 37 children were evaluated. All presented cerebral calcifications on the initial scan, predominantly at cortical-white matter junction. At follow-up the calcifications had diminished in number, size, or density, or a combination in 34 of the children (92%, 95% confidence interval 79% to 97%), were no longer visible in one child, and remained unchanged in two children. No child showed an increase in calcifications. The calcifications at the cortical-white matter junction which were no longer visible at follow-up occurred predominately in the parietal and occipital lobes. These imaging changes were not associated with any clear clinical improvements.Conclusion The detection of cerebral calcifications should not be considered a major criterion for late diagnosis of congenital Zika syndrome, nor should the absence of calcifications be used to exclude the diagnosis.

Diffuse periventricular calcification and brain atrophy: A case of neonatal central nervous system cytomegalovirus infection.

TORCH refers to the most common congenitally acquired infections: toxoplasma, rubella, cytomegalovirus, and herpes simplex virus. Neonatal cytomegalovirus infection remains a common cause of congenital infection worldwide with effects ranging from hearing impairment to significant neurological morbidity. We report a case of a term neonate with ventriculomegaly on prenatal ultrasound who presented with low birth weight, small head circumference, hepatosplenomegaly, and purpuric rash on physical exam. Central nervous system cytomegalovirus infection typically shows periventricular calcifications and associated deep white matter damage and ventriculomegaly. Ultrasound, computed tomography, and magnetic resonance imaging have different roles in the diagnosis of congenital central nervous system cytomegalovirus infection. Many imaging features of congenital cytomegalovirus are distinctive, and can spur a diagnostic work-up as well as help provide a prognosis.

Cyclosporine A Treatment Inhibits Abcc6-Dependent Cardiac Necrosis and Calcification following Coxsackievirus B3 Infection in Mice.

Coxsackievirus type B3 (CVB3) is a cardiotropic enterovirus. Infection causes cardiomyocyte necrosis and myocardial inflammation. The damaged tissue that results is replaced with fibrotic or calcified tissue, which can lead to permanently altered cardiac function. The extent of pathogenesis among individuals exposed to CVB3 is dictated by a combination of host genetics, viral virulence, and the environment. Here, we aimed to identify genes that modulate cardiopathology following CVB3 infection. 129S1 mice infected with CVB3 developed increased cardiac pathology compared to 129X1 substrain mice despite no difference in viral burden. Linkage analysis identified a major locus on chromosome 7 (LOD: 8.307, P<0.0001) that controlled the severity of cardiac calcification and necrosis following infection. Sub-phenotyping and genetic complementation assays identified Abcc6 as the underlying gene. Microarray expression profiling identified genotype-dependent regulation of genes associated with mitochondria. Electron microscopy examination showed elevated deposition of hydroxyapatite-like material in the mitochondrial matrices of infected Abcc6 knockout (Abcc6-/-) mice but not in wildtype littermates. Cyclosporine A (CsA) inhibits mitochondrial permeability transition pore opening by inhibiting cyclophilin D (CypD). Treatment of Abcc6 -/- mice with CsA reduced cardiac necrosis and calcification by more than half. Furthermore, CsA had no effect on the CVB3-induced phenotype of doubly deficient CypD-/-Abcc6-/- mice. Altogether, our work demonstrates that mutations in Abcc6 render mice more susceptible to cardiac calcification following CVB3 infection. Moreover, we implicate CypD in the control of cardiac necrosis and calcification in Abcc6-deficient mice, whereby CypD inhibition is required for cardioprotection.

The prospective association of Chlamydia pneumoniae and four other pathogens with development of coronary artery calcium: the multi-ethnic study of atherosclerosis (MESA).

OBJECTIVE: Previous basic and cross-sectional studies obtained conflicting results regarding the association of pathogens with coronary artery calcium (CAC). The aim of this study is to prospectively evaluate this association in a population-based cohort. METHODS: We examined 5744 individuals aged 45-84 years at baseline (2000-02) who underwent repeated CAC assessment on average 2.4 years later (a half at visit 2 [2002-04] and the other half at visit 3 [2004-05]). CAC incidence was defined as newly detectable CAC at follow-up (475 cases of 2942 participants). CAC progression was defined as annualized change in CAC Agatston score ≥10 units/year if baseline CAC score >0 to <100 or ≥10%/year if baseline score ≥ 100 (1537 cases of 2802 participants). Seropositivity was assessed in the entire cohort for Chlamydia pneumoniae and in a random sample (n = 873) for Helicobacter pylori, cytomegalovirus, herpes simplex virus, and hepatitis A virus. RESULTS: Seropositivity to C. pneumoniae was not significantly associated with CAC incidence (odds ratio [OR] 1.11 [95% CI, 0.88-1.39], P = 0.371) or progression (1.14 [0.96-1.36], P = 0.135) even in unadjusted models. When CAC incidence and progression were combined, we observed significant association with C. pneumoniae seropositivity before adjustment (OR 1.17 [1.03-1.33], P = 0.016) but not in a model adjusting for traditional risk factors (1.04 [0.90-1.19], P = 0.611). The results were consistent across subgroups according to age, sex, and race/ethnicity. None of five pathogens or their accrual was associated with CAC incidence and progression in the subsample. CONCLUSION: Our prospective study does not support the pathophysiological involvement of these pathogens in CAC development.

α-Lipoic acid attenuates coxsackievirus B3-induced ectopic calcification in heart, pancreas, and lung.

Ectopic mineralization of soft tissues is known to be a typical response to systemic imbalance of various metabolic factors as well as tissue injury, leading to severe clinical consequences. In this study, coxsackievirus B3 (CVB3) infection in mice resulted in significant tissue injury, especially in the heart and pancreas. Inflammatory damage and apoptotic cell death were observed in CVB3-infected heart and pancreas tissues. Along with tissue damage, substantial ectopic calcification was detected in CVB3-infected heart, pancreas, and lung tissues, as determined by von Kossa staining and calcium content quantification. In addition, CVB3 infection induced upregulation of osteogenic signals, including six genes (BMP2, SPARC, Runx2, osteopontin, collagen type I, and osterix) in the heart, three genes (SPARC, osteopontin, and collagen type I) in the pancreas, and two genes (BMP2 and alkaline phosphatase) in the lung, as determined by quantitative real-time PCR analysis. Intriguingly, we showed that α-lipoic acid diminished CVB3-mediated inflammatory and apoptotic tissue damage, subsequently ameliorating ectopic calcification via the suppression of osteogenic signals. Collectively, our data provide evidence that ectopic calcification induced by CVB3 infection is implicated in the induction of osteogenic propensity, and α-lipoic acid may be a potential therapeutic agent to ameliorate pathologic calcification.

Targeting of the osteoclastogenic RANKL-RANK axis prevents osteoporotic bone loss and soft tissue calcification in coxsackievirus B3-infected mice.

Bone mineralization is a normal physiological process, whereas ectopic calcification of soft tissues is a pathological process that leads to irreversible tissue damage. We have established a coxsackievirus B3 (CVB3)-infected mouse model that manifests both osteoporosis and ectopic calcification specifically in heart, pancreas, and lung. The CVB3-infected mice showed increased serum concentrations of both cytokines including IL-1ß, TNF-α, and the receptor activator of NF-κB ligand (RANKL) that stimulate osteoclast formation and of the osteoclast-derived protein tartrate-resistant acid phosphatase 5b. They exhibited more osteoclasts in bone, with no change in the number of osteoblasts, and a decrease in bone formation and the serum concentration of osteoblast-produced osteocalcin. These results indicate that CVB3-induced osteoporosis is likely due to upregulation of osteoclast formation and function, in addition to decreased osteoblast activity. In addition, the serum in the CVB3-infected mice contained a high inorganic phosphate content, which causes ectopic calcification. RANKL treatment induced an increase in the in vitro cardiac fibroblast calcification by inorganic phosphate via the upregulation of osteogenic BMP2, SPARC, Runx2, Fra-1, and NF-κB signaling. We finally observed that i.p. administration of RANK-Fc, a recombinant antagonist of RANKL, prevented bone loss as well as ectopic calcification in CVB3-infected mice. Thus, our results indicate that RANKL may contribute to both abnormal calcium deposition in soft tissues and calcium depletion in bone. In addition, our animal model should provide a tool for the development of new therapeutic agents for calcium disturbance in soft and hard tissues.

Central nervous system complications and neuroradiological findings in children with chronic active Epstein-Barr virus infection.

BACKGROUND: Although many neurological complications have been described in acute Epstein-Barr virus infection, few reports have discussed the central nervous system complications in chronic active Epstein-Barr virus (CAEBV) infection. METHODS: We retrospectively surveyed the medical records of 14 patients with CAEBV infection in our institute. Neuroradiological studies were performed in 10 of these patients. RESULTS: Five had no neurological symptoms, whereas two presented with posterior reversible encephalopathy syndrome, one presented with basal ganglia calcification, and one presented with falx cerebri hemorrhage. Although both of the posterior reversible encephalopathy syndrome cases developed epilepsy several years after recovering from prolonged neurological deterioration, the others had no neurological sequelae. CONCLUSIONS: This study revealed that various central nervous system complications may occur during the clinical course in pediatric CAEBV patients.

Epstein-Barr virus-positive lymphoproliferative disorder associated with old organized chronic subdural hematoma.

This report describes a case of an immunocompetent 77-year-old male with Epstein-Barr virus (EBV)-positive lymphoproliferative disorder associated with calcified chronic subdural hematoma (CSH). On the day prior to consultation in our outpatient clinic, the patient fell from his bed, striking his frontal head on the floor. Magnetic resonance imaging showed ill-defined lesions in the right frontal-temporal subdural regions. At surgery, a hard and thickened outer membrane of a CSH and muddy organized subdural hematoma were observed. However, macroscopic neoplastic lesions were not apparent. Histologically, there were atypical lymphoid cells scattered or conglomerated in some areas of the thick outer membrane of the CSH. They were composed of occasional large atypical lymphoid cells. The lesions were accompanied by necrosis. Atypical lymphoid cells were immunopositive for B-cell markers but not for T-cell markers. EBNA2 was seen in the nuclei of tumor cells. Atypical lymphoid cells showed positive signals for EBV-encoded small RNAs (EBERs) on in situ hybridization. These findings were consistent with EBV-positive lymphoproliferative disorder associated with CSH. These results also suggested that EBV and the inflammatory reaction found in the CSH could be the etiological factors in the pathogenesis of lymphoproliferative disorder.

Clinical and neuroimaging profile of HIV-1 encephalopathy in infancy and childhood in a sub-Saharan African country.

BACKGROUND: Neurological dysfunction in AIDS is common, occurring in as many as eighty percent of children. Thus, it is important to recognize the central nervous system imaging appearance of HIV, in particular those of HIV encephalopathy, as this is an AIDS defining illness and with distinct neuro-imaging features essential for early diagnosis and timely therapeutic intervention AIM: To identify the clinical features in HIV-1 infection of the central nervous system and their associated neuroradiological correlates. METHODS: Retrospective review of the records of all children with HIV-1 encephalopathy identified among children with neurological and developmental problems and who were on follow up at a child development and neurology clinic in an African city. RESULTS: A total of 22 children (10 male and 12 female) with HIV-1 encephalopathy were identified among 2382 children with various forms of neurological and developmental problems and who were on follow up at a child development and neurology clinic for a little bit over eight years period. All the children acquired the infection vertically. The age range of these children was between 10 months to 14 years. The median age was 5.6 years. The mean duration of symptom was 3.2 years. Global delay or regression in development along with signs of pyramidal tract involvement and seizures were the commonest clinical signs observed in these children. Neuro-behavioral problems were commonly observed among preschool and school aged children. In older children and preadolescents focal seizures with or with out neurologic deficit and neuroradiological findings were common. Nonhemorrhagic stroke was rare and occurred in one child and another child had cortical blindness. Three children had no neurological deficit. Rapid progression of the disease carried grave prognosis. Opportunistic infections and tumors of the central nervous system were also uncommon among these children. Brain volume loss with dilatation of the lateral ventricle, bilateral symmetrical or asymmetrical calcification of the basal ganglia and periventricular involvement of the white matter were the commonest neuro-radiological findings observed in these children. CONCLUSION: Atrophy of the brain with dilatation of the lateral ventricles and calcification of the basal ganglia and peri-ventricular involvement of the white matter were the commonest neuro-radiological findings in children with HIV-1 encephalopathy. Similarly global delay or regression in development along with pyramidal tract signs and seizures were the commonest neurological findings. Behavioral problems were common in preschool and school aged children. Focal seizures were common in older children and preadolescents. Rapid progression of the disease carried grave prognosis.

Asymmetric dimethylarginine and coronary artery calcium scores are increased in patients infected with human immunodeficiency virus.

OBJECTIVE: Patients infected with human immunodeficiency virus (HIV) have an increased risk for cardiovascular events and mortality. Elevated concentrations of asymmetric dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase, are associated with increased subclinical atherosclerosis and cardiovascular events. The objective of this study was to determine whether plasma ADMA levels are increased in patients infected with HIV and whether this is associated with cardiovascular risk factors, inflammatory/thrombotic biomarkers, and elevated coronary artery calcium scores (CACS). METHODS: HIV-infected patients and control patients were recruited in a case-control study. Medical history and laboratory measurements including plasma ADMA and biomarkers for inflammation and thrombosis such as C-reactive protein (CRP), fibrinogen, and homocysteine were obtained in both cohorts. Using multidetector computed tomography, CACS were measured in Agatston Units (AU). Bivariate differences between HIV-infected and control patients were analyzed. RESULTS: HIV-infected patients (n=37, male=27, age=45 years) had significantly higher concentrations of ADMA (0.40±0.10 µmol/l) compared to a similarly matched cohort of non-HIV-infected patients (n=43, male=27, age=45 years), (0.35±0.07 µmol/l, p=0.03). There were no significant differences in CRP, homocysteine, and fibrinogen between the two cohorts. However, HIV-infected patients had a higher CACS distribution compared to control patients [0.0 (8.5) vs. 0.0 (0.0) AU, p=0.01]. In a multivariable regression analysis HIV-infected patients with a relative CACS of 75-90% for age and gender had the highest ADMA concentrations (0.48±0.09 µmol/l, p=0.04) among all CACS subgroups. CONCLUSION: HIV-infected patients have significantly higher ADMA concentrations compared to control patients. In addition, increased CACS was associated with elevated ADMA concentrations. Thus, increased ADMA levels appear to be associated with the presence of subclinical atherosclerosis in HIV-infected patients.

Demonstration of Chlamydophila pneumoniae, Mycoplasma pneumoniae, Cytomegalovirus, and Epstein-Barr virus in atherosclerotic coronary arteries, nonrheumatic calcific aortic and rheumatic stenotic mitral valves by polymerase chain reaction.

OBJECTIVE: The aim of this study was to investigate whether bacterial and viral infectious agents can be demonstrated in atherosclerotic lesions of patients with coronary artery disease (CAD) as well as in stenotic aortic and mitral valves from patients undergoing heart valve replacement. METHODS: In this cross-sectional study, the presence of Chlamydophila pneumoniae, Mycoplasma pneumoniae, Cytomegalovirus (CMV), and Epstein-Barr virus (EBV) was investigated by polymerase chain reaction in atherosclerotic and non-atherosclerotic vascular samples taken from patients undergoing coronary artery bypass surgery due to CAD, and from patients undergoing aortic (AVR) and/or mitral valve replacement (MVR) secondary to valvular stenosis. For statistical analyses ANOVA, Chi-square test or Fisher's exact test were used. RESULTS: The presence of C. pneumoniae, M. pneumoniae, and CMV in atherosclerotic versus non-atherosclerotic samples was as follows: 30% vs. 16.7% (p=0.222), 6.7% vs. 3.3% (p=0.554), and 10% vs. 0% (p=0.076), respectively. In valve group, same pathogens were present in AVR and MVR patients as follows: 24.2% vs. 21.4% (p=0.773), 9.1% vs. 7.1% (p=0.758), and 21.2% vs. 11.9% (p=0.275). EBV DNA was not detected in any of vascular specimens, but in one (3%) patient with AVR (p=0.256). CONCLUSION: Our results suggest that C. pneumoniae, M. pneumoniae, and CMV are present with similar frequency both in atherosclerotic and non-atherosclerotic vessels. We conclude that although non-atherosclerotic, vascular samples of CAD patients are invaded by infectious agents as like as atherosclerotic vessels. We further conclude that C. pneumoniae, M. pneumoniae, and CMV are present in stenotic aortic and mitral valves and atherosclerotic tissues with similar frequency indicating that atherosclerosis and valvular stenosis might share a common etiology related to infection.

In vivo characterization of cytokine profiles and viral load during murine cytomegalovirus-induced acute myocarditis.

BACKGROUND: Murine cytomegalovirus (MCMV) is an etiologic agent of acute and chronic myocarditis in BALB/c mice. Immunologic host responses appear to play a key role in pathogenesis but have been incompletely defined. METHODS: BALB/c mice were infected with a sublethal dose of MCMV. Cytokine transcription and viral load (measured by quantitative real-time polymerase chain reaction) and histopathological analyses were performed at specified time points. RESULTS: Increased tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and interferon (IFN)-gamma, as well as IL-10 mRNA transcripts, were detected in the hearts of infected mice starting at Day 1 post-infection (p.i.), with peak levels occurring at Day 8 p.i. (7-fold, 14-fold, 41-fold, and 16-fold higher than background, respectively). Peak cytokine transcription significantly correlated with a 10-fold increase in viral load (P<.001) at Day 8 p.i. Myocarditis-related pathological changes, measured by infiltration foci, were greatest at Day 8 p.i., corresponding with peak cytokine transcription and significantly correlated with IFN-gamma levels (P<.0001). Infiltration foci were predominantly composed of CD3(+) T cells. Cardiac calcification was observed in most infected mice predominantly over the right ventricle. Histological analysis of heart sections from mice infected with recombinant enhanced green fluorescence protein-MCMV revealed a localized and sporadic pattern of virus throughout all heart layers. CONCLUSIONS: MCMV-induced myocarditis in BALB/c mice is characterized by in vivo production of proinflammatory cytokines in a pattern correlating with MCMV viral load. The infection pattern and inflammatory response is highly localized, sporadic, and involves endocardium, epicardium, as well as the myocardium, with greatest amounts of virus detected in areas of pathologic calcification.

HIV positivity, protease inhibitor exposure and subclinical atherosclerosis: a systematic review and meta-analysis of observational studies.

CONTEXT: Patients with HIV may have increased risk of atherosclerotic cardiovascular disease owing to multiple biological mechanisms. OBJECTIVE: To evaluate the evidence for subclinical atherosclerosis among patients with HIV. DESIGN: Systematic review of observational studies. DATA SOURCES: We searched Medline, Cochrane DSR, ACP Journal Club, DARE, CMR, HTA, NHSEED, Embase and the Cochrane Controlled Trials Register for studies published before November 2008. STUDY SELECTION: Eligible studies were cross-sectional, cohort, or case-control studies reporting carotid ultrasound intima-media thickness (CIMT), focal plaque incidence, or coronary artery calcium (CAC), as determined by HIV positivity or protease inhibitor (PI) exposure. DATA EXTRACTION: Two independent reviewers abstracted data using a standardised form. The primary outcome was weighted mean difference (WMD) for CIMT comparing HIV positive versus negative patients. Other outcomes included WMD by PI exposure and the odds ratio (OR) for a focal carotid plaque or CAC. Data from six cross-sectional, seven case-control and 13 cohort studies were included, involving 5456 HIV positive and 3600 HIV negative patients. RESULTS: The weighted mean CIMT was 0.04 mm thicker among patients with HIV than among non-HIV patients (95% CI 0.02 to 0.06; p<0.001). HIV positivity was not associated with carotid plaque or CAC. PI exposure did not significantly affect CIMT, carotid plaque, or CAC. There was evidence of publication bias and stratified analysis and meta-regression showed outcomes were influenced by study design, age, gender and smoking. However, HIV positivity slightly increased CIMT even after sensitivity analyses. CONCLUSIONS: HIV infection and PI exposure are not strong independent risk factors for subclinical atherosclerosis. Confounding may contribute to overestimation of the risk associated with HIV and PI exposure.

Local gene transfer to calcified tissue cells using prolonged infusion of a lentiviral vector.

Gene transfer using viral vectors offers the potential for the sustained expression of proteins in specific target tissues. However, in the case of calcified tissues, in vivo delivery remains problematic because of limited accessibility. The aim of this study was to test the efficiency of lentiviral vectors (LVs) on osteogenic cells in vitro, and determine the feasibility of directly transducing resident bone cells in vivo. LVs encoding for green fluorescent protein (GFP) and ameloblastin (AMBN), a protein associated with mineralization not reported in bone, were generated. The transduction efficiency of the LVs was evaluated using the MC3T3 cell line and primary calvaria-derived osteogenic cells. For in vivo delivery, the LVs were infused using osmotic minipumps through holes created in the bone of the rat hemimandible and tibia. The production of GFP and AMBN in vitro and in vivo was monitored using fluorescence microscopy. Both transgenes were expressed in MC3T3 and primary osteogenic cells. In vivo, GFP was detected at the infusion site and fibroblast-like cells, osteoblasts, osteocytes and osteoclasts expressed AMBN. Our data demonstrate, for the first time, that primary osteogenic cells are efficiently transduced with LVs and that their infusion is advantageous for locally delivering DNA to bone cells.