Development of a new blood test for the diagnosis of the allergies to dental metals

Congrès du GIRSO, Lille, avril 2011

Differential inhibition of human immunodeficiency virus type 1 in peripheral blood mononuclear cells and TZM-bl cells by endotoxin-mediated chemokine and gamma interferon production.

Bacterial lipopolysaccharide (endotoxin) is a frequent contaminant of biological specimens and is also known to be a potent inducer of beta-chemokines and other soluble factors that inhibit HIV-1 infection in vitro. Though lipopolysaccharide (LPS) has been shown to stimulate the production of soluble HIV-1 inhibitors in cultures of monocyte-derived macrophages, the ability of LPS to induce similar inhibitors in other cell types is poorly characterized. Here we show that LPS exhibits potent anti-HIV activity in phytohemagglutinin-stimulated peripheral blood mononuclear cells (PBMCs) but has no detectable anti-HIV-1 activity in TZM-bl cells. The anti-HIV-1 activity of LPS in PBMCs was strongly associated with the production of beta-chemokines from CD14-positive monocytes. Culture supernatants from LPS-stimulated PBMCs exhibited potent anti-HIV-1 activity when added to TZM-bl cells but, in this case, the antiviral activity appeared to be related to IFN-gamma rather than to beta-chemokines. These observations indicate that LPS stimulates PBMCs to produce a complex array of soluble HIV-1 inhibitors, including beta-chemokines and IFN-gamma, that differentially inhibit HIV-1 depending on the target cell type. The results also highlight the need to use endotoxin-free specimens to avoid artifacts when assessing HIV-1-specific neutralizing antibodies in PBMC-based assays.

The implementation of a translational study involving a primary care based behavioral program to improve blood pressure control: The HTN-IMPROVE study protocol (01295).

BACKGROUND: Despite the impact of hypertension and widely accepted target values for blood pressure (BP), interventions to improve BP control have had limited success. OBJECTIVES: We describe the design of a 'translational' study that examines the implementation, impact, sustainability, and cost of an evidence-based nurse-delivered tailored behavioral self-management intervention to improve BP control as it moves from a research context to healthcare delivery. The study addresses four specific aims: assess the implementation of an evidence-based behavioral self-management intervention to improve BP levels; evaluate the clinical impact of the intervention as it is implemented; assess organizational factors associated with the sustainability of the intervention; and assess the cost of implementing and sustaining the intervention. METHODS: The project involves three geographically diverse VA intervention facilities and nine control sites. We first conduct an evaluation of barriers and facilitators for implementing the intervention at intervention sites. We examine the impact of the intervention by comparing 12-month pre/post changes in BP control between patients in intervention sites versus patients in the matched control sites. Next, we examine the sustainability of the intervention and organizational factors facilitating or hindering the sustained implementation. Finally, we examine the costs of intervention implementation. Key outcomes are acceptability and costs of the program, as well as changes in BP. Outcomes will be assessed using mixed methods (e.g., qualitative analyses--pattern matching; quantitative methods--linear mixed models). DISCUSSION: The study results will provide information about the challenges and costs to implement and sustain the intervention, and what clinical impact can be expected.

Altered gene expression and DNA damage in peripheral blood cells from Friedreich's ataxia patients: cellular model of pathology.

The neurodegenerative disease Friedreich's ataxia (FRDA) is the most common autosomal-recessively inherited ataxia and is caused by a GAA triplet repeat expansion in the first intron of the frataxin gene. In this disease, transcription of frataxin, a mitochondrial protein involved in iron homeostasis, is impaired, resulting in a significant reduction in mRNA and protein levels. Global gene expression analysis was performed in peripheral blood samples from FRDA patients as compared to controls, which suggested altered expression patterns pertaining to genotoxic stress. We then confirmed the presence of genotoxic DNA damage by using a gene-specific quantitative PCR assay and discovered an increase in both mitochondrial and nuclear DNA damage in the blood of these patients (p<0.0001, respectively). Additionally, frataxin mRNA levels correlated with age of onset of disease and displayed unique sets of gene alterations involved in immune response, oxidative phosphorylation, and protein synthesis. Many of the key pathways observed by transcription profiling were downregulated, and we believe these data suggest that patients with prolonged frataxin deficiency undergo a systemic survival response to chronic genotoxic stress and consequent DNA damage detectable in blood. In conclusion, our results yield insight into the nature and progression of FRDA, as well as possible therapeutic approaches. Furthermore, the identification of potential biomarkers, including the DNA damage found in peripheral blood, may have predictive value in future clinical trials.

Diagnosis of partial body radiation exposure in mice using peripheral blood gene expression profiles.

In the event of a terrorist-mediated attack in the United States using radiological or improvised nuclear weapons, it is expected that hundreds of thousands of people could be exposed to life-threatening levels of ionizing radiation. We have recently shown that genome-wide expression analysis of the peripheral blood (PB) can generate gene expression profiles that can predict radiation exposure and distinguish the dose level of exposure following total body irradiation (TBI). However, in the event a radiation-mass casualty scenario, many victims will have heterogeneous exposure due to partial shielding and it is unknown whether PB gene expression profiles would be useful in predicting the status of partially irradiated individuals. Here, we identified gene expression profiles in the PB that were characteristic of anterior hemibody-, posterior hemibody- and single limb-irradiation at 0.5 Gy, 2 Gy and 10 Gy in C57Bl6 mice. These PB signatures predicted the radiation status of partially irradiated mice with a high level of accuracy (range 79-100%) compared to non-irradiated mice. Interestingly, PB signatures of partial body irradiation were poorly predictive of radiation status by site of injury (range 16-43%), suggesting that the PB molecular response to partial body irradiation was anatomic site specific. Importantly, PB gene signatures generated from TBI-treated mice failed completely to predict the radiation status of partially irradiated animals or non-irradiated controls. These data demonstrate that partial body irradiation, even to a single limb, generates a characteristic PB signature of radiation injury and thus may necessitate the use of multiple signatures, both partial body and total body, to accurately assess the status of an individual exposed to radiation.

Altered ultrasonic vocalization and impaired learning and memory in Angelman syndrome mouse model with a large maternal deletion from Ube3a to Gabrb3.

Angelman syndrome (AS) is a neurobehavioral disorder associated with mental retardation, absence of language development, characteristic electroencephalography (EEG) abnormalities and epilepsy, happy disposition, movement or balance disorders, and autistic behaviors. The molecular defects underlying AS are heterogeneous, including large maternal deletions of chromosome 15q11-q13 (70%), paternal uniparental disomy (UPD) of chromosome 15 (5%), imprinting mutations (rare), and mutations in the E6-AP ubiquitin ligase gene UBE3A (15%). Although patients with UBE3A mutations have a wide spectrum of neurological phenotypes, their features are usually milder than AS patients with deletions of 15q11-q13. Using a chromosomal engineering strategy, we generated mutant mice with a 1.6-Mb chromosomal deletion from Ube3a to Gabrb3, which inactivated the Ube3a and Gabrb3 genes and deleted the Atp10a gene. Homozygous deletion mutant mice died in the perinatal period due to a cleft palate resulting from the null mutation in Gabrb3 gene. Mice with a maternal deletion (m-/p+) were viable and did not have any obvious developmental defects. Expression analysis of the maternal and paternal deletion mice confirmed that the Ube3a gene is maternally expressed in brain, and showed that the Atp10a and Gabrb3 genes are biallelically expressed in all brain sub-regions studied. Maternal (m-/p+), but not paternal (m+/p-), deletion mice had increased spontaneous seizure activity and abnormal EEG. Extensive behavioral analyses revealed significant impairment in motor function, learning and memory tasks, and anxiety-related measures assayed in the light-dark box in maternal deletion but not paternal deletion mice. Ultrasonic vocalization (USV) recording in newborns revealed that maternal deletion pups emitted significantly more USVs than wild-type littermates. The increased USV in maternal deletion mice suggests abnormal signaling behavior between mothers and pups that may reflect abnormal communication behaviors in human AS patients. Thus, mutant mice with a maternal deletion from Ube3a to Gabrb3 provide an AS mouse model that is molecularly more similar to the contiguous gene deletion form of AS in humans than mice with Ube3a mutation alone. These mice will be valuable for future comparative studies to mice with maternal deficiency of Ube3a alone.

Association of a peripheral blood metabolic profile with coronary artery disease and risk of subsequent cardiovascular events.

BACKGROUND: Molecular tools may provide insight into cardiovascular risk. We assessed whether metabolites discriminate coronary artery disease (CAD) and predict risk of cardiovascular events. METHODS AND RESULTS: We performed mass-spectrometry-based profiling of 69 metabolites in subjects from the CATHGEN biorepository. To evaluate discriminative capabilities of metabolites for CAD, 2 groups were profiled: 174 CAD cases and 174 sex/race-matched controls ("initial"), and 140 CAD cases and 140 controls ("replication"). To evaluate the capability of metabolites to predict cardiovascular events, cases were combined ("event" group); of these, 74 experienced death/myocardial infarction during follow-up. A third independent group was profiled ("event-replication" group; n=63 cases with cardiovascular events, 66 controls). Analysis included principal-components analysis, linear regression, and Cox proportional hazards. Two principal components analysis-derived factors were associated with CAD: 1 comprising branched-chain amino acid metabolites (factor 4, initial P=0.002, replication P=0.01), and 1 comprising urea cycle metabolites (factor 9, initial P=0.0004, replication P=0.01). In multivariable regression, these factors were independently associated with CAD in initial (factor 4, odds ratio [OR], 1.36; 95% CI, 1.06 to 1.74; P=0.02; factor 9, OR, 0.67; 95% CI, 0.52 to 0.87; P=0.003) and replication (factor 4, OR, 1.43; 95% CI, 1.07 to 1.91; P=0.02; factor 9, OR, 0.66; 95% CI, 0.48 to 0.91; P=0.01) groups. A factor composed of dicarboxylacylcarnitines predicted death/myocardial infarction (event group hazard ratio 2.17; 95% CI, 1.23 to 3.84; P=0.007) and was associated with cardiovascular events in the event-replication group (OR, 1.52; 95% CI, 1.08 to 2.14; P=0.01). CONCLUSIONS: Metabolite profiles are associated with CAD and subsequent cardiovascular events.

Patterns in blood pressure medication use in US incident dialysis patients over the first 6 months.

BACKGROUND: Several observational studies have evaluated the effect of a single exposure window with blood pressure (BP) medications on outcomes in incident dialysis patients, but whether BP medication prescription patterns remain stable or a single exposure window design is adequate to evaluate effect on outcomes is unclear. METHODS: We described patterns of BP medication prescription over 6 months after dialysis initiation in hemodialysis and peritoneal dialysis patients, stratified by cardiovascular comorbidity, diabetes, and other patient characteristics. The cohort included 13,072 adult patients (12,159 hemodialysis, 913 peritoneal dialysis) who initiated dialysis in Dialysis Clinic, Inc., facilities January 1, 2003-June 30, 2008, and remained on the original modality for at least 6 months. We evaluated monthly patterns in BP medication prescription over 6 months and at 12 and 24 months after initiation. RESULTS: Prescription patterns varied by dialysis modality over the first 6 months; substantial proportions of patients with prescriptions for beta-blockers, renin angiotensin system agents, and dihydropyridine calcium channel blockers in month 6 no longer had prescriptions for these medications by month 24. Prescription of specific medication classes varied by comorbidity, race/ethnicity, and age, but little by sex. The mean number of medications was 2.5 at month 6 in hemodialysis and peritoneal dialysis cohorts. CONCLUSIONS: This study evaluates BP medication patterns in both hemodialysis and peritoneal dialysis patients over the first 6 months of dialysis. Our findings highlight the challenges of assessing comparative effectiveness of a single BP medication class in dialysis patients. Longitudinal designs should be used to account for changes in BP medication management over time, and designs that incorporate common combinations should be considered.

Vascular access, fluid resuscitation, and blood transfusion in pediatric trauma.

Trauma care in the general population has largely become protocol-driven, with an emphasis on fast and efficient treatment, good team communication at all levels of care including prehospital care, initial resuscitation, intensive care, and rehabilitation. Most available literature on trauma care has focused on adults, allowing the potential to apply concepts from adult care to pediatric care. But there remain issues that will always be specific to pediatric patients that may not translate from adults. Several new devices such as intraosseous (IO) needle systems and techniques such as ultrasonography to cannulate central and peripheral veins have become available for integration into our pre-existing trauma care system for children. This review will focus specifically on the latest techniques and evidence available for establishing intravenous access, rational approaches to fluid resuscitation, and blood product transfusion in the pediatric trauma patient.

Maternal rank influences the outcome of aggressive interactions between immature chimpanzees

© 2014 The Association for the Study of Animal Behaviour.For many long-lived mammalian species, extended maternal investment has a profound effect on offspring integration in complex social environments. One component of this investment may be aiding young in aggressive interactions, which can set the stage for offspring social position later in life. Here we examined maternal effects on dyadic aggressive interactions between immature (<12 years) chimpanzees. Specifically, we tested whether relative maternal rank predicted the probability of winning an aggressive interaction. We also examined maternal responses to aggressive interactions to determine whether maternal interventions explain interaction outcomes. Using a 12-year behavioural data set (2000-2011) from Gombe National Park, Tanzania, we found that relative maternal rank predicted the probability of winning aggressive interactions in male-male and male-female aggressive interactions: offspring were more likely to win if their mother outranked their opponent's mother. Female-female aggressive interactions occurred infrequently (two interactions), so could not be analysed. The probability of winning was also higher for relatively older individuals in male-male interactions, and for males in male-female interactions. Maternal interventions were rare (7.3% of 137 interactions), suggesting that direct involvement does not explain the outcome for the vast majority of aggressive interactions. These findings provide important insight into the ontogeny of aggressive behaviour and early dominance relationships in wild apes and highlight a potential social advantage for offspring of higher-ranking mothers. This advantage may be particularly pronounced for sons, given male philopatry in chimpanzees and the potential for social status early in life to translate more directly to adult rank.

Monitoring maternal Beta carotene and retinol consumption may decrease the incidence of neurodevelopmental disorders in offspring.

Retinoic acids (13-cis and 13-trans) are known teratogens, and their precursor is retinol, a form of vitamin A. In 1995, Rothman et al demonstrated an association between excessive vitamin A, >10,000 IU/day, during the first trimester of pregnancy and teratogenic effects, particularly in the central nervous system. However, vitamin A deficiency has long been known to be deleterious to the mother and fetus. Therefore, there may be a narrow therapeutic ratio for vitamin A during pregnancy that has not previously been fully appreciated. Neurodevelopmental disorders may not be apparent by macroscopic brain examination or imaging, and proving the existence of a behavioral teratogen is not straightforward. However, an excess of retinoic acid and some neurodevelopmental disorders are both associated with abnormalities in cerebellar morphology. Physical and chemical evidence strongly supports the notion that beta carotene crosses the placenta and is metabolized to retinol. Only very limited amounts of beta carotene are stored in fetal fat cells as evidenced by the fact that maternal fat is yellow from beta carotene, whereas non-brown neonatal fat is white. Furthermore, newborns of carotenemic mothers do not share the yellow complexion of their mothers. The excess 13-trans retinoic acid derived from metabolized beta carotene in the fetus increases the concentration of the more teratogenic 13-cis retinoic acid since the isomerization equilibrium is shifted to the left. Therefore, this paper proposes that consideration be given to monitoring all potential sources of fetal 13-cis and 13-trans retinoic acid, including nutritional supplements, dietary retinol, and beta carotene, particularly in the first trimester of pregnancy.

CD45+ Cells Present Within Mesenchymal Stem Cell Populations Affect Network Formation of Blood-Derived Endothelial Outgrowth Cells.

Mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) represent promising cell sources for angiogenic therapies. There are, however, conflicting reports regarding the ability of MSCs to support network formation of endothelial cells. The goal of this study was to assess the ability of human bone marrow-derived MSCs to support network formation of endothelial outgrowth cells (EOCs) derived from umbilical cord blood EPCs. We hypothesized that upon in vitro coculture, MSCs and EOCs promote a microenvironment conducive for EOC network formation without the addition of angiogenic growth supplements. EOC networks formed by coculture with MSCs underwent regression and cell loss by day 10 with a near 4-fold and 2-fold reduction in branch points and mean segment length, respectively, in comparison with networks formed by coculture vascular smooth muscle cell (SMC) cocultures. EOC network regression in MSC cocultures was not caused by lack of vascular endothelial growth factor (VEGF)-A or changes in TGF-β1 or Ang-2 supernatant concentrations in comparison with SMC cocultures. Removal of CD45+ cells from MSCs improved EOC network formation through a 2-fold increase in total segment length and number of branch points in comparison to unsorted MSCs by day 6. These improvements, however, were not sustained by day 10. CD45 expression in MSC cocultures correlated with EOC network regression with a 5-fold increase between day 6 and day 10 of culture. The addition of supplemental growth factors VEGF, fibroblastic growth factor-2, EGF, hydrocortisone, insulin growth factor-1, ascorbic acid, and heparin to MSC cocultures promoted stable EOC network formation over 2 weeks in vitro, without affecting CD45 expression, as evidenced by a lack of significant differences in total segment length (p=0.96). These findings demonstrate the ability of MSCs to support EOC network formation correlates with removal of CD45+ cells and improves upon the addition of soluble growth factors.

The relationship between blood IL-12p40 level and melanoma progression

© 2014 UICC.Cytokines such as Interleukin (IL)212p70 ("IL-12") and IL-23 can influence tumor progression. We tested the hypothesis that blood levels of IL-12p40, the common subunit of both cytokines, are associated with melanoma progression. Blood from 2,048 white melanoma patients were collected at a single institution between March 1998 and March 2011. Plasma levels of IL-12p40 were determined for 573 patients (discovery), 249 patients (Validation 1) and 244 patients (Validation 2). Per 10-unit change of IL-12p40 level was used to investigate associations with melanoma patient outcome among all patients or among patients with early or advanced stage. Among stage I/II melanoma patients in the pooled data set, after adjustment for sex, age, stage and blood draw time from diagnosis, elevated IL-12p40 was associated with melanoma recurrence [hazard ratio (HR)51.04 per 10-unit increase in IL-12p40, 95% CI 1.02-1.06, p58.48 × 10-5]; Elevated IL-12p40 was also associated with a poorer melanoma specific survival (HR51.06, 95% CI 1.03-1.09, p53.35 × 10-5) and overall survival (HR51.05, 95% CI 1.03-1.08, p58.78 × 10-7) in multivariate analysis. Among stage III/IV melanoma patients in the pooled data set, no significant association was detected between elevated IL-12p40 and overall survival, or with melanoma specific survival, with or without adjustment for the above covariates. Early stage melanoma patients with elevated IL-12p40 levels are more likely to develop disease recurrence and have a poorer survival. Further investigation with a larger sample size will be needed to determine the role of IL-12p40 in advanced stage melanoma patients.

Association of Common Genetic Polymorphisms with Melanoma Patient IL-12p40 Blood Levels, Risk, and Outcomes.

Recent investigation has identified association of IL-12p40 blood levels with melanoma recurrence and patient survival. No studies have investigated associations of single-nucleotide polymorphisms (SNPs) with melanoma patient IL-12p40 blood levels or their potential contributions to melanoma susceptibility or patient outcome. In the current study, 818,237 SNPs were available for 1,804 melanoma cases and 1,026 controls. IL-12p40 blood levels were assessed among 573 cases (discovery), 249 cases (case validation), and 299 controls (control validation). SNPs were evaluated for association with log[IL-12p40] levels in the discovery data set and replicated in two validation data sets, and significant SNPs were assessed for association with melanoma susceptibility and patient outcomes. The most significant SNP associated with log[IL-12p40] was in the IL-12B gene region (rs6897260, combined P=9.26 × 10(-38)); this single variant explained 13.1% of variability in log[IL-12p40]. The most significant SNP in EBF1 was rs6895454 (combined P=2.24 × 10(-9)). A marker in IL12B was associated with melanoma susceptibility (rs3213119, multivariate P=0.0499; OR=1.50, 95% CI 1.00-2.24), whereas a marker in EBF1 was associated with melanoma-specific survival in advanced-stage patients (rs10515789, multivariate P=0.02; HR=1.93, 95% CI 1.11-3.35). Both EBF1 and IL12B strongly regulate IL-12p40 blood levels, and IL-12p40 polymorphisms may contribute to melanoma susceptibility and influence patient outcome.

Menthol attenuates respiratory irritation and elevates blood cotinine in cigarette smoke exposed mice.

Addition of menthol to cigarettes may be associated with increased initiation of smoking. The potential mechanisms underlying this association are not known. Menthol, likely due to its effects on cold-sensing peripheral sensory neurons, is known to inhibit the sensation of irritation elicited by respiratory irritants. However, it remains unclear whether menthol modulates cigarette smoke irritancy and nicotine absorption during initial exposures to cigarettes, thereby facilitating smoking initiation. Using plethysmography in a C57Bl/6J mouse model, we examined the effects of L-menthol, the menthol isomer added to cigarettes, on the respiratory sensory irritation response to primary smoke irritants (acrolein and cyclohexanone) and smoke of Kentucky reference 2R4 cigarettes. We also studied L-menthol's effect on blood levels of the nicotine metabolite, cotinine, immediately after exposure to cigarette smoke. L-menthol suppressed the irritation response to acrolein with an apparent IC₅₀ of 4 ppm. Suppression was observed even at acrolein levels well above those necessary to produce a maximal response. Cigarette smoke, at exposure levels of 10 mg/m³ or higher, caused an immediate and marked sensory irritation response in mice. This response was significantly suppressed by L-menthol even at smoke concentrations as high as 300 mg/m³. Counterirritation by L-menthol was abolished by treatment with a selective inhibitor of Transient Receptor Potential Melastatin 8 (TRPM8), the neuronal cold/menthol receptor. Inclusion of menthol in the cigarette smoke resulted in roughly a 1.5-fold increase in plasma cotinine levels over those observed in mice exposed to smoke without added menthol. These findings document that, L-menthol, through TRPM8, is a strong suppressor of respiratory irritation responses, even during highly noxious exposures to cigarette smoke or smoke irritants, and increases blood cotinine. Therefore, L-menthol, as a cigarette additive, may promote smoking initiation and nicotine addiction.