Placental growth factor contributes to micro-vascular abnormalization and blood-retinal barrier breakdown in diabetic retinopathy.

OBJECTIVE: There are controversies regarding the pro-angiogenic activity of placental growth factor (PGF) in diabetic retinopathy (DR). For a better understanding of its role on the retina, we have evaluated the effect of a sustained PGF over-expression in rat ocular media, using ciliary muscle electrotransfer (ET) of a plasmid encoding rat PGF-1 (pVAX2-rPGF-1). MATERIALS AND METHODS: pVAX2-rPGF-1 ET in the ciliary muscle (200 V/cm) was achieved in non diabetic and diabetic rat eyes. Control eyes received saline or naked plasmid ET. Clinical follow up was carried out over three months using slit lamp examination and fluorescein angiography. After the control of rPGF-1 expression, PGF-induced effects on retinal vasculature and on the blood-external barrier were evaluated respectively by lectin and occludin staining on flat-mounts. Ocular structures were visualized through histological analysis. RESULTS: After fifteen days of rPGF-1 over-expression in normal eyes, tortuous and dilated capillaries were observed. At one month, microaneurysms and moderate vascular sprouts were detected in mid retinal periphery in vivo and on retinal flat-mounts. At later stages, retinal pigmented epithelial cells demonstrated morphological abnormalities and junction ruptures. In diabetic retinas, PGF expression rose between 2 and 5 months, and, one month after ET, rPGF-1 over-expression induced glial activation and proliferation. CONCLUSION: This is the first demonstration that sustained intraocular PGF production induces vascular and retinal changes similar to those observed in the early stages of diabetic retinopathy. PGF and its receptor Flt-1 may therefore be looked upon as a potential regulatory target at this stage of the disease.

Umbilical cord blood transplantation from unrelated donors in patients with Philadelphia chromosome-positive acute lymphoblastic leukemia.

There are very few disease-specific studies focusing on outcomes of umbilical cord blood transplantation for Philadelphia chromosome-positive acute lymphoblastic leukemia. We report the outcome of 45 patients with Philadelphia chromosome-positive acute lymphoblastic leukemia who underwent myeloablative single unit cord blood transplantation from unrelated donors within the GETH/GITMO cooperative group. Conditioning regimens were based on combinations of thiotepa, busulfan, cyclophospamide or fludarabine, and antithymocyte globulin. At the time of transplantation, 35 patients (78%) were in first complete remission, four (8%) in second complete remission and six (14%) in third or subsequent response. The cumulative incidence of myeloid engraftment was 96% at a median time of 20 days and significantly better for patients receiving higher doses of CD34(+) cells. The incidence of acute grade II-IV graft-versus-host disease was 31%, while that of overall chronic graft-versus-host disease was 53%. Treatment-related mortality was 17% at day +100 and 31% at 5 years. The 5-year relapse, event-free survival and overall survival rates were 31%, 36% and 44%, respectively. Although the event-free and overall survival rates in patients without BCR/ABL transcripts detectable at time of transplant were better than those in whom BCR/ABL transcripts were detected (46% versus 24% and 60% versus 30%, respectively) these differences were not statistically significant in the univariate analysis (P=0.07). These results demonstrate that umbilical cord blood transplantation from unrelated donors can be a curative treatment for a substantial number of patients with Philadelphia chromosome-positive acute lymphoblastic leukemia.

Clinicopathologic analysis of acute myeloid leukemia arising from chronic myelomonocytic leukemia.

Acute myeloid leukemia arising from chronic myelomonocytic leukemia is currently classified as acute myeloid leukemia with myelodysplasia-related changes, a high-risk subtype. However, the specific features of these cases have not been well described. We studied 38 patients with chronic myelomonocytic leukemia who progressed to acute myeloid leukemia. We compared the clinicopathologic and genetic features of these cases with 180 patients with de novo acute myeloid leukemia and 34 patients with acute myeloid leukemia following myelodysplastic syndromes. We also examined features associated with progression from chronic myelomonocytic leukemia to acute myeloid leukemia by comparing the progressed chronic myelomonocytic leukemia cases with a cohort of chronic myelomonocytic leukemia cases that did not transform to acute myeloid leukemia. Higher white blood cell count, marrow cellularity, karyotype risk score, and Revised International Prognostic Scoring System score were associated with more rapid progression from chronic myelomonocytic leukemia to acute myeloid leukemia. Patients with acute myeloid leukemia ex chronic myelomonocytic leukemia were older (P<0.01) and less likely to receive aggressive treatment (P=0.02) than de novo acute myeloid leukemia patients. Most cases showed monocytic differentiation and fell into the intermediate acute myeloid leukemia karyotype risk group; 55% had normal karyotype and 17% had NPM1 mutation. Median overall survival was 6 months, which was inferior to de novo acute myeloid leukemia (17 months, P=0.002) but similar to post myelodysplastic syndrome acute myeloid leukemia. On multivariate analysis of all acute myeloid leukemia patients, only age and karyotype were independent prognostic variables for overall survival. Our findings indicate that acute myeloid leukemia following chronic myelomonocytic leukemia displays aggressive behavior and support placement of these cases within the category of acute myeloid leukemia with myelodysplasia-related changes. The poor prognosis of these patients may be related to an older population and lack of favorable-prognosis karyotypes that characterize many de novo acute myeloid leukemia cases.

Response to Infliximab in Crohn's Disease: Genetic Analysis Supporting Expression Profile.

Substantial proportion of Crohn's disease (CD) patients shows no response or a limited response to treatment with infliximab (IFX) and to identify biomarkers of response would be of great clinical and economic benefit. The expression profile of five genes (S100A8-S100A9, G0S2, TNFAIP6, and IL11) reportedly predicted response to IFX and we aimed at investigating their etiologic role through genetic association analysis. Patients with active CD (350) who received at least three induction doses of IFX were included and classified according to IFX response. A tagging strategy was used to select genetic polymorphisms that cover the variability present in the chromosomal regions encoding the identified genes with altered expression. Following genotyping, differences between responders and nonresponders to IFX were observed in haplotypes of the studied regions: S100A8-S100A9 (rs11205276* G/rs3014866* C/rs724781* C/rs3006488* A; P = 0.05); G0S2 (rs4844486* A/rs1473683* T; P = 0.15); TNFAIP6 (rs11677200* C/rs2342910* A/rs3755480* G/rs10432475* A; P = 0.10); and IL11 (rs1126760* C/rs1042506* G; P = 0.07). These differences were amplified in patients with colonic and ileocolonic location for all but the TNFAIP6 haplotype, which evidenced significant difference in ileal CD patients. Our results support the role of the reported expression signature as predictive of anti-TNF outcome in CD patients and suggest an etiological role of those top-five genes in the IFX response pathway.

Incremental Prognostic Value of Myocardial Blood Flow Quantification with Rb-82 Cardiac PET in Patients With Known or Suspected CAD

Rb-82cardiac PET has been used to non-invasively assess myocardial blood flow (MBF)and myocardial flow reserve (MFR). The impact of MBF and MFR for predictingmajor adverse cardiovascular events (MACE) has not been investigated in aprospective study, which was our aim. MATERIAL AND METHODS: In total, 280patients (65±10y, 36% women) with known or suspected CAD were prospectivelyenrolled. They all underwent both a rest and adenosine stress Rb-82 cardiacPET/CT. Dynamic acquisitions were processed with the FlowQuant 2.1.3 softwareand analyzed semi-quantitatively (SSS, SDS) and quantitatively (MBF, MFR) andreported using the 17-segment AHA model. Patients were stratified based on SDS,stress MBF and MFR and allocated into tertiles. For each group, annualizedevent rates were computed by dividing the number of annualized MACE (cardiacdeath, myocardial infarction, revascularisation or hospitalisation forcardiac-related event) by the sum of individual follow-up periods in years.Outcome were analysed for each group using Kaplan-Meier event-free survivalcurves and compared using the log-rank test. Multivariate analysis wasperformed in a stepwise fashion using Cox proportional hazards regressionmodels (p<0.05 for model inclusion). RESULTS: In a median follow-up of 256days (range 168-440d), 44 MACE were observed. Ischemia (SDS≥2) was observed in95 patients who had higher annualized MACE rate as compared to those without(55% vs. 9.8%, p<0.0001). The group with the lowest MFR tertile (MFR<1.76)had higher MACE rate than the two highest tertiles (51% vs. 9% and 14%,p<0.0001). Similarly, the group with the lowest stress MBF tertile(MBF<1.78mL/min/g) had the highest annualized MACE rate (41% vs. 26% and 6%,p=0.0002). On multivariate analysis, the addition of MFR or stress MBF to SDSsignificantly increased the global χ2 (from 56 to 60, p=0.04; and from56 to 63, p=0.01). The best prognostic power was obtained in a model combiningSDS (p<0.001) and stress MBF (p=0.01). Interestingly, the integration ofstress MBF enhanced risk stratification even in absence of ischemia.CONCLUSIONS: Quantification of MBF or MFR in Rb-82 cardiac PET/CT providesindependent and incremental prognostic information over semi-quantitativeassessment with SDS and is of value for risk stratification.

Modelling the cardiovascular system for automatic interpretation of the blood pressure curve

A four compartment model of the cardiovascular system is developed. To allow for easy interpretation and to minimise the number of parameters, an effort was made to keep the model as simple as possible. A sensitivity analysis is first carried out to determine which are the most important model parameters to characterise the blood pressure signal. A four stage process is then described which accurately determines all parameter values. This process is applied to data from three patients and good agreement is shown in all cases.

Integrative analysis of RUNX1 downstream pathways and target genes.

BACKGROUND: The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia. RESULTS: Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFbeta, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFbeta. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes. CONCLUSION: This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications

Quantitative sequence analysis of FBN1 premature termination codons provides evidence for incomplete NMD in leukocytes.

We improved, evaluated, and used Sanger sequencing for quantification of single nucleotide polymorphism (SNP) variants in transcripts and gDNA samples. This improved assay resulted in highly reproducible relative allele frequencies (e.g., for a heterozygous gDNA 50.0+/-1.4%, and for a missense mutation-bearing transcript 46.9+/-3.7%) with a lower detection limit of 3-9%. It provided excellent accuracy and linear correlation between expected and observed relative allele frequencies. This sequencing assay, which can also be used for the quantification of copy number variations (CNVs), methylations, mosaicisms, and DNA pools, enabled us to analyze transcripts of the FBN1 gene in fibroblasts and blood samples of patients with suspected Marfan syndrome not only qualitatively but also quantitatively. We report a total of 18 novel and 19 known FBN1 sequence variants leading to a premature termination codon (PTC), 26 of which we analyzed by quantitative sequencing both at gDNA and cDNA levels. The relative amounts of PTC-containing FBN1 transcripts in fresh and PAXgene-stabilized blood samples were significantly higher (33.0+/-3.9% to 80.0+/-7.2%) than those detected in affected fibroblasts with inhibition of nonsense-mediated mRNA decay (NMD) (11.0+/-2.1% to 25.0+/-1.8%), whereas in fibroblasts without NMD inhibition no mutant alleles could be detected. These results provide evidence for incomplete NMD in leukocytes and have particular importance for RNA-based analyses not only in FBN1 but also in other genes.

Estimating arterial blood gases with the EABC® System (“Earlobe Arterialized Blood Collector”) in critically ill patients. A validation study.

The Earlobe Arterialized Blood Collector® is a minimally invasive system able to perform arterialized capillary blood gas analysis from the earlobe (EL). A prospective validation study was performed in 55 critical ill patients. Sampling failure rate was high (53.6%). Risk factors were age > 65 years, diabetes, vasoactive drug therapy and noradrenaline (NA) doses above 0.22 μg / kg / min. Multivariate analysis showed age > 65 years was the only factor independently associated with failure. Concordance analysis with arterial blood gases and Bland-Altman agreement evaluation were insufficient for validating the new system for all gasometrical variables.

Using 80 kVp versus 120 kVp in perfusion CT measurement of regional cerebral blood flow.

Perfusion CT studies of regional cerebral blood flow (rCBF), involving sequential acquisition of cerebral CT sections during IV contrast material administration, have classically been reported to be achieved at 120 kVp. We hypothesized that using 80 kVp should result in the same image quality while significantly lowering the patient's radiation dose, and we evaluated this assumption. In five patients undergoing cerebral CT survey, one section level was imaged at 120 kVp and 80 kVp, before and after IV administration of iodinated contrast material. These four cerebral CT sections obtained in each patient were analyzed with special interest to contrast, noise, and radiation dose. Contrast enhancement at 80 kVp is significantly increased (P < .001), as well as contrast between gray matter and white matter after contrast enhancement (P < .001). Mean noise at 80 kVp is not statistically different (P = .042). Finally, performance of perfusion CT studies at 80 kVp, keeping mAs constant, lowers the radiation dose by a factor of 2.8. We, thus, conclude that 80 kVp acquisition of perfusion CT studies of rCBF will result in increased contrast enhancement and should improve rCBF analysis, with a reduced patient's irradiation.

Blood plasma lipidomic signature of epicardial fat in healthy obese women.

OBJECTIVES: A lipidomic approach was employed in a clinically well-defined cohort of healthy obese women to explore blood lipidome phenotype ascribed to body fat deposition, with emphasis on epicardial adipose tissue (EAT). METHODS: The present investigation delivered a lipidomics signature of epicardial adiposity under healthy clinical conditions using a cohort of 40 obese females (age: 25-45 years, BMI: 28-40 kg/m(2) ) not showing any metabolic disease traits. Lipidomics analysis of blood plasma was employed in combination with in vivo quantitation of mediastinal fat depots by computerized tomography. RESULTS: All cardiac fat depots correlated to indicators of hepatic dysfunctions (ALAT and ASAT), which describe physiological connections between hepatic and cardiac steatosis. Plasma lipidomics encompassed overall levels of lipid classes, fatty acid profiles, and individual lipid species. EAT and visceral fat associated with diacylglycerols (DAG), triglycerides, and distinct phospholipid and sphingolipid species. A pattern of DAG and phosphoglycerols was specific to EAT. CONCLUSIONS: Human blood plasma lipidomics appears to be a promising clinical and potentially diagnostic readout for patient stratification and monitoring. Association of blood lipidomics signature to regio-specific mediastinal and visceral adiposity under healthy clinical conditions may help provide more biological insights into obese patient stratification for cardiovascular disease risks.

Blood pressure changes in acute ischemic stroke and outcome with respect to stroke etiology.

OBJECTIVE: Previous research suggested that proper blood pressure (BP) management in acute stroke may need to take into account the underlying etiology. METHODS: All patients with acute ischemic stroke registered in the ASTRAL registry between 2003 and 2009 were analyzed. Unfavorable outcome was defined as modified Rankin Scale score >2. A local polynomial surface algorithm was used to assess the effect of baseline and 24- to 48-hour systolic BP (SBP) and mean arterial pressure (MAP) on outcome in patients with lacunar, atherosclerotic, and cardioembolic stroke. RESULTS: A total of 791 patients were included in the analysis. For lacunar and atherosclerotic strokes, there was no difference in the predicted probability of unfavorable outcome between patients with an admission BP of <140 mm Hg, 140-160 mm Hg, or >160 mm Hg (15.3 vs 12.1% vs 20.8%, respectively, for lacunar, p = 015; 41.0% vs 41.5% vs 45.5%, respectively, for atherosclerotic, p = 075), or between patients with BP increase vs decrease at 24-48 hours (18.7% vs 18.0%, respectively, for lacunar, p = 0.84; 43.4% vs 43.6%, respectively, for atherosclerotic, p = 0.88). For cardioembolic strokes, increase of BP at 24-48 hours was associated with higher probability of unfavorable outcome compared to BP reduction (53.4% vs 42.2%, respectively, p = 0.037). Also, the predicted probability of unfavorable outcome was significantly different between patients with an admission BP of <140 mm Hg, 140-160 mm Hg, and >160 mm Hg (34.8% vs 42.3% vs 52.4%, respectively, p < 0.01). CONCLUSIONS: This study provides evidence to support that BP management in acute stroke may have to be tailored with respect to the underlying etiopathogenetic mechanism.

Inactivation des pathogènes des produits sanguins labiles: entre enjeux financiers et conséquences possibles à long terme [Pathogen reduction of blood components: from financial issues to possible long-term consequences].

Pathogen inactivation of blood products represents a global and major paradigm shift in transfusion medicine. In the next near future, it is likely that most blood products will be inactivated by various physicochemical approaches. The concept of blood safety will be challenged as well as transfusion medicine practice, notably for donor selection or biological qualification. In this context, it seems mandatory to develop analytical economic approaches by assessing costs-benefits ratio of blood transfusion as well as to set up cohorts of patients based on hemovigilance networks allowing rigorous scientific analysis of the benefits and the risks of blood transfusion at short- and long-term.

Correlation between vasoconstrictor roles and mRNA expression of alpha1-adrenoceptor subtypes in blood vessels of genetically engineered mice.

We examined the contribution of each alpha(1)-adrenoceptor (AR) subtype in noradrenaline (NAd)-evoked contraction in the thoracic aortas and mesenteric arteries of mice. Compared with the concentration-response curves (CRCs) for NAd in the thoracic aortas of wild-type (WT) mice, the CRCs of mutant mice showed a significantly lower sensitivity. The pD(2) value in rank order is as follows: WT mice (8.21) > alpha(1B)-adrenoceptor knockout (alpha(1B)-KO) (7.77) > alpha(1D)-AR knockout (alpha(1D)-KO) (6.44) > alpha(1B)- and alpha(1D)-AR double knockout (alpha(1BD)-KO) (5.15). In the mesenteric artery, CRCs for NAd did not differ significantly between either WT (6.52) and alpha(1B)-KO mice (7.12) or alpha(1D)-KO (6.19) and alpha(1BD)-KO (6.29) mice. However, the CRC maximum responses to NAd in alpha(1D)- and alpha(1BD)-KO mice were significantly lower than those in WT and alpha(1B)-KO mice. Except in the thoracic aortas of alpha(1BD)-KO mice, the competitive antagonist prazosin inhibited the contraction response to NAd with high affinity. However, prazosin produced shallow Schild slopes in the vessels of mice lacking the alpha(1D)-AR gene. In the thoracic aorta, pA(2) values in WT mice for KMD-3213 and BMY7378 were 8.25 and 8.46, respectively, and in alpha(1B)-KO mice they were 8.49 and 9.13, respectively. In the mesenteric artery, pA(2) values in WT mice for KMD-3213 and BMY7378 were 8.34 and 7.47, respectively, and in alpha(1B)-KO mice they were 8.11 and 7.82, respectively. These pharmacological findings were in fairly good agreement with findings from comparison of CRCs, with the exception of the mesenteric arteries of WT and alpha(1B)-KO mice, which showed low affinities to BMY7378. We performed a quantitative analysis of the mRNA expression of each alpha(1)-AR subtype in these vessels in order to examine the correlation between mRNA expression level and the predominance of each alpha(1)-AR subtype in mediating vascular contraction. The rank order of each alpha(1)-AR subtype in terms of its vasoconstrictor role was in fairly good agreement with the level of expression of mRNA of each subtype, that is, alpha(1D)-AR > alpha(1B)-AR > alpha(1A)-AR in the thoracic aorta and alpha(1D)-AR > alpha(1A)-AR > alpha(1B)-AR in the mesenteric artery. No dramatic compensatory change of alpha(1)-AR subtype in mutant mice was observed in pharmacological or quantitative mRNA expression analysis.

Proteomic analysis of mononuclear cells of patients with minimal-change nephrotic syndrome of childhood.

Background/Aims. Recently, peripheral blood mononuclear cell transcriptome analysis has identified genes that are upregulated in relapsing minimal-change nephrotic syndrome (MCNS). In order to investigate protein expression in peripheral blood mononuclear cells (PBMC) from relapsing MCNS patients, we performed proteomic comparisons of PBMC from patients with MCNS in relapse and controls. METHODS: PBMC from a total of 20 patients were analysed. PBMC were taken from five patients with relapsing MCNS, four in remission, five patients with other glomerular diseases and six controls. Two dimensional electrophoresis was performed and proteome patterns were compared. RESULTS: Automatic heuristic clustering analysis allowed us to pool correctly the gels from the MCNS patients in the relapse and in the control groups. Using hierarchical population matching, nine spots were found to be increased in PBMC from MCNS patients in relapse. Four spots were identified by mass spectrometry. Three of the four proteins identified (L-plastin, alpha-tropomyosin and annexin III) were cytoskeletal-associated proteins. Using western blot and immunochemistry, L-plastin and alpha-tropomyosin 3 concentrations were found to be enhanced in PBMC from MCNS patients in relapse. Conclusions. These data indicate that a specific proteomic profile characterizes PBMC from MCNS patients in relapse. Proteins involved in PBMC cytoskeletal rearrangement are increased in relapsing MCNS. We hypothesize that T-cell cytoskeletal rearrangement may play a role in the pathogenesis of MCNS by altering the expression of cell surface receptors and by modifying the interaction of these cells with glomerular cells.