A Biochemical Approach to Understand the Pathogenesis of Advanced Pulmonary Arterial Hypertension: Metabolomic Profiles of Arginine,Sphingosine-1-Phosphate,and Heme of Human Lung

Pulmonary arterial hypertension (PAH) is a vascular disease characterized by persistent precapillary pulmonary hypertension (PH), leading to progressive right heart failure and premature death. The pathological mechanisms underlying this condition remain elusive. Analysis of global metabolomics from lung tissue of patients with PAH (n = 8) and control lung tissue (n = 8) leads to a better understanding of disease progression. Using a combination of high-throughput liquid-and-gas-chromatography-based mass spectrometry, we showed unbiased metabolomic profiles of disrupted arginine pathways with increased Nitric oxide (NO) and decreased arginine. Our results also showed specific metabolic pathways and genetic profiles with increased Sphingosine-1-phosphate (S1P) metabolites as well as increased Heme metabolites with altered oxidative pathways in the advanced stage of the human PAH lung. The results suggest that PAH has specific metabolic pathways contributing to the vascular remodeling in severe pulmonary hypertension. Profiling metabolomic alterations of the PAH lung has provided a new understanding of the pathogenic mechanisms of PAH, which benefits therapeutic targeting to specific metabolic pathways involved in the progression of PAH.     

Childhood Learning Disabilities and Atypical Dementia: A Retrospective Chart Review

Objective

To further our understanding of the association between self-reported childhood learning disabilities (LDs) and atypical dementia phenotypes (Atypical Dementia), including logopenic primary progressive aphasia (L-PPA), Posterior Cortical Atrophy (PCA), and Dysexecutive-type Alzheimer’s Disease (AD).

Methods

This retrospective case series analysis of 678 comprehensive neuropsychological assessments compared rates of self-reported LD between dementia patients diagnosed with Typical AD and those diagnosed with Atypical Dementia. 105 cases with neuroimaging or CSF data available and at least one neurology follow-up were identified as having been diagnosed by the neuropsychologist with any form of neurodegenerative dementia. These cases were subject to a consensus diagnostic process among three dementia experts using validated clinical criteria for AD and PPA. LD was considered Probable if two or more statements consistent with prior LD were documented within the Social & Developmental History of the initial neuropsychological evaluation.

Results

85 subjects (Typical AD n=68, Atypical AD n=17) were included in the final analysis. In logistic regression models adjusted for age, gender, handedness, education and symptom duration, patients with Probable LD, compared to patients without Probable LD, were significantly more likely to be diagnosed with Atypical Dementia vs. Typical AD (OR 13.1, 95% CI 1.3-128.4). All three of the L-PPA cases reporting a childhood LD endorsed childhood difficulty with language. By contrast, both PCA cases reporting Probable childhood LD endorsed difficulty with attention and/or math.

Conclusions

In people who develop dementia, childhood LD may predispose to atypical phenotypes. Future studies are required to confirm whether atypical neurodevelopment predisposes to regional-specific neuropathology in AD and other dementias.     

Reproducibility of In Vivo Corneal Confocal Microscopy Using an Automated Analysis Program for Detection of Diabetic Sensorimotor Polyneuropathy

Objective

In vivo Corneal Confocal Microscopy (IVCCM) is a validated, non-invasive test for diabetic sensorimotor polyneuropathy (DSP) detection, but its utility is limited by the image analysis time and expertise required. We aimed to determine the inter- and intra-observer reproducibility of a novel automated analysis program compared to manual analysis.

Methods

In a cross-sectional diagnostic study, 20 non-diabetes controls (mean age 41.4±17.3y, HbA1c 5.5±0.4%) and 26 participants with type 1 diabetes (42.8±16.9y, 8.0±1.9%) underwent two separate IVCCM examinations by one observer and a third by an independent observer. Along with nerve density and branch density, corneal nerve fibre length (CNFL) was obtained by manual analysis (CNFL_MANUAL), a protocol in which images were manually selected for automated analysis (CNFL_{SEMI-AUTOMATED}), and one in which selection and analysis were performed electronically (CNFL_{FULLY-AUTOMATED}). Reproducibility of each protocol was determined using intraclass correlation coefficients (ICC) and, as a secondary objective, the method of Bland and Altman was used to explore agreement between protocols.

Results

Mean CNFL_Manual was 16.7±4.0, 13.9±4.2 mm/mm² for non-diabetes controls and diabetes participants, while CNFL_{Semi-Automated} was 10.2±3.3, 8.6±3.0 mm/mm² and CNFL_{Fully-Automated} was 12.5±2.8, 10.9 ± 2.9 mm/mm². Inter-observer ICC and 95% confidence intervals (95%CI) were 0.73(0.56, 0.84), 0.75(0.59, 0.85), and 0.78(0.63, 0.87), respectively (p = NS for all comparisons). Intra-observer ICC and 95%CI were 0.72(0.55, 0.83), 0.74(0.57, 0.85), and 0.84(0.73, 0.91), respectively (p<0.05 for CNFL_{Fully-Automated} compared to others). The other IVCCM parameters had substantially lower ICC compared to those for CNFL. CNFL_{Semi-Automated} and CNFL_{Fully-Automated} underestimated CNFL_Manual by mean and 95%CI of 35.1(-4.5, 67.5)% and 21.0(-21.6, 46.1)%, respectively.

Conclusions

Despite an apparent measurement (underestimation) bias in comparison to the manual strategy of image analysis, fully-automated analysis preserves CNFL reproducibility. Future work must determine the diagnostic thresholds specific to the fully-automated measure of CNFL.     

Role of Cbl-PI3K Interaction during Skeletal Remodeling in a Murine Model of Bone Repair

Mice in which Cbl is unable to bind PI3K (YF mice) display increased bone volume due to enhanced bone formation and repressed bone resorption during normal bone homeostasis. We investigated the effects of disrupted Cbl-PI3K interaction on fracture healing to determine whether this interaction has an effect on bone repair. Mid-diaphyseal femoral fractures induced in wild type (WT) and YF mice were temporally evaluated via micro-computed tomography scans, biomechanical testing, histological and histomorphometric analyses. Imaging analyses revealed no change in soft callus formation, increased bony callus formation, and delayed callus remodeling in YF mice compared to WT mice. Histomorphometric analyses showed significantly increased osteoblast surface per bone surface and osteoclast numbers in the calluses of YF fractured mice, as well as increased incorporation of dynamic bone labels. Furthermore, using laser capture micro-dissection of the fracture callus we found that cells lacking Cbl-PI3K interaction have higher expression of Osterix, TRAP, and Cathepsin K. We also found increased expression of genes involved in propagating PI3K signaling in cells isolated from the YF fracture callus, suggesting that the lack of Cbl-PI3K interaction perhaps results in enhanced PI3K signaling, leading to increased bone formation, but delayed remodeling in the healing femora.     

New clinical and experimental insights into Old World and neotropical ocular toxoplasmosis

Retinal lesions or other ocular manifestations are serious consequences of infection with the protozoan parasite Toxoplasma gondii. Whilst classically considered a consequence of congenital transmission, recent screening studies estimated that 2% of T. gondii seropositive persons in Europe and North America have retinal lesions, most of them persisting unnoticed. The situation is more dramatic in South America, probably due to the predominance of virulent strains. Some of these strains seem to exhibit ocular or neuronal tropism and are responsible for severe ocular lesions. Despite the medical importance, the physiopathological mechanisms have only recently begun to be elucidated. The particular immune-privileged situation in the eye has to be considered. Studies on French patients showed low or undetectable ocular parasite loads, but a clear Th1/Th17 type immune reaction. Suitable mouse models have appeared in the last few years. Using such a model, IL-17A proved to impair parasite control and induce pathology. In contrast, in South American patients, the parasite seems to be much less efficiently controlled through a Th2 type or suppressive immune response that favors parasite replication. Finally, several host genetic markers controlling immune response factors have been associated with ocular involvement of T. gondii infection, mainly in South America.     

Scaling with body mass of mitochondrial respiration from the white muscle of three phylogenetically, morphologically and behaviorally disparate teleost fishes

White muscle (WM) fibers in many fishes often increase in size from <50 μm in juveniles to >250 μm in adults. This leads to increases in intracellular diffusion distances that may impact the scaling with body mass of muscle metabolism. We have previously found similar negative scaling of aerobic capacity (mitochondrial volume density, V _mt) and the rate of an aerobic process (post-contractile phosphocreatine recovery) in fish WM. In the present study, we examined the scaling with body mass of oxygen consumption rates of isolated mitochondria (VO_2mt) from WM in three species from different families that vary in morphology and behavior: an active, pelagic species (bluefish, Pomatomus saltatrix), a relatively inactive demersal species (black sea bass, Centropristis striata), and a sedentary, benthic species (southern flounder, Paralichthys lethostigma). In contrast to our prior studies, the measurement of respiration in isolated mitochondria is not influenced by the diffusion of oxygen or metabolites. V _mt was measured in WM and in high-density isolates used for VO_2mt measurements. WM V _mt was significantly higher in the bluefish than in the other two species and VO_2mt was independent of body mass when expressed per milligram protein or per milliliter mitochondria. The size-independence of VO_2mt indicates that differences in WM aerobic function result from variation in V _mt and not to changes in VO_2mt. This is consistent with our prior work that indicated that while diffusion constraints influence mitochondrial distribution, the negative scaling of aerobic processes like post-contractile PCr recovery can largely be attributed to the body size dependence of V _mt.     

High expression and activity of ecto-5′-nucleotidase/CD73 in the male murine reproductive tract

Extracellular ATP and its hydrolysis product adenosine modulate various reproductive functions such as those requiring contraction, steroidogenesis, and maintenance of fluid composition. Interestingly, adenosine might act as a key capacitative effector for mammalian spermatozoa to acquire the capacity for fertilisation. Extracellular nucleotide levels are affected by cell surface ectonucleotidases, amongst which the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family regroups the most abundant and effective enzymes to hydrolyse ATP and ADP to AMP in physiological conditions. In the male reproductive tract three members of this family have been indentified: NTPDase1, NTPDase2 and NTPDase3 (Martín-Satué et al. in Histochem Cell Biol 131:615–628, 2009). The purpose of the present study was to characterize in the male reproductive tract the expression profile of the main enzyme responsible for the generation of adenosine from AMP, namely the ecto-5′-nucleotidase (CD73). The enzyme was identified by immunological techniques and by in situ enzymatic assays, including inhibition experiments with α,β-methylene-ADP, a specific CD73 inhibitor. High levels of ecto-5′-nucleotidase were detected in testes in association with both germinal and somatic cells, in smooth muscle cells throughout the tract, in secretory epithelia from exocrine glands, and remarkably, in principal cells of epididymis, where co-localization with NTPDase3 was found. The relevance of this co-expression on nucleotide hydrolysis in these cells directly involved in the control of sperm fluid composition was addressed biochemically. This study suggests close regulation of extracellular nucleoside and nucleotide levels in the genital tract by ecto-5′-nucleotidase that, in concurrence with NTPDases, may impact male fertility.     

Identification of potent and reversible cruzipain inhibitors for the treatment of Chagas disease

Identification of potent and reversible cruzipain inhibitors for the treatment of Chagas disease is described. The identified inhibitors bearing an amino nitrile warhead in P1 exhibit low nanomolar in vitro potency against cruzipain. Further SAR in P2 portion led to the identification of compounds, such as 26, that have a unique selectivity profile against other cysteine proteases and offering new opportunities for safer treatment of Chagas disease.