Visualization of incrementally learned projection trajectories for longitudinal data.

Longitudinal studies that continuously generate data enable the capture of temporal variations in experimentally observed parameters, facilitating the interpretation of results in a time-aware manner. We propose IL-VIS (incrementally learned visualizer), a new machine learning pipeline that incrementally learns and visualizes a progression trajectory representing the longitudinal changes in longitudinal studies. At each sampling time point in an experiment, IL-VIS generates a snapshot of the longitudinal process on the data observed thus far, a new feature that is beyond the reach of classical static models. We first verify the utility and correctness of IL-VIS using simulated data, for which the true progression trajectories are known. We find that it accurately captures and visualizes the trends and (dis)similarities between high-dimensional progression trajectories. We then apply IL-VIS to longitudinal multi-electrode array data from brain cortical organoids when exposed to different levels of quinolinic acid, a metabolite contributing to many neuroinflammatory diseases including Alzheimer's disease, and its blocking antibody. We uncover valuable insights into the organoids' electrophysiological maturation and response patterns over time under these conditions.

Stability Studies of Kynurenine Pathway Metabolites in Blood Components Define Optimal Blood Processing Conditions.

The kynurenine pathway (KP) is the main pathway of tryptophan (TRP) metabolism that generates energy for multiple cellular processes. The activity of this pathway has been shown to be dysregulated in multiple human diseases. The resultant modulation of metabolites has been suggested to comprise biomarkers to track disease progression or could identify new therapeutic targets. While metabolite changes can be measured readily in blood, there is limited knowledge on the effect of blood matrices and sample processing time may have on the stability of KP metabolites. Understanding the stability of KP metabolites in blood is integral to obtaining accurate KP data to correlate with clinical pathology. Hence, the aim of this study was to assess the concentration of KP metabolites in matched whole blood, plasma and serum. The impact of pre-analytical sample processing time in the various blood matrices was also analysed. Serum and plasma had the higher concentration of KP metabolites compared to whole blood. Furthermore, concentrations of KP metabolites declined when the collected blood was processed after 24 hours storage at 4°C. Our study shows that that type of blood matrix and the time to processing have an impact on the stability of the KP metabolites. Serum or plasma are the preferred choice of matrix and the isolation of these matrices from whole blood is best performed immediately after collection for optimal analytical KP data.

The kynurenine pathway relates to post-acute COVID-19 objective cognitive impairment and PASC.

OBJECTIVE: To determine the prevalence and natural history of post-acute COVID-19 objective cognitive impairment and function, and their relationship to demographic, clinical factors, post-acute sequelae of COVID-19 (PASC), and biomarkers. METHODS: A total of 128 post-acute COVID-19 patients (age = 46 ± 15; 42% women, acute disease severity: not hospitalized: 38.6% mild: 0-1 symptoms, 52% 2+ symptoms; 9.4% hospitalized) completed standard cognition, olfaction, and mental health examinations 2-, 4-, and 12-month post diagnosis. Over the same time frame, WHO-defined PASC was determined. Blood cytokines, peripheral neurobiomarkers, and kynurenine pathway (KP) metabolites were measured. Objective cognitive function was demographically/practice corrected, and impairment prevalence was determined using the evidence-based Global Deficit Score method to detect at least mild cognitive impairment (GDS > 0.5). Linear mixed effect regression models with time effect (month post diagnosis) evaluated the relationships to cognition. RESULTS: Across the 12-month study period, mild to moderate cognitive impairment ranged from 16% to 26%, and 46.5% were impaired at least once. Impairment associated with poorer work capacity (p < 0.05), and 2-month objectively tested anosmia (p < 0.05). PASC with (p = 0.01) and without disability (p < 0.03) associated with acute COVID-19 severity. KP measures showed prolonged activation (2 to 8 months) (p < 0.0001) linked to IFN-beta in those with PASC. Of the blood analytes, only the KP metabolites (elevated quinolinic acid, 3-hydroxyanthranilic acid, kynurenine, the kynurenine/tryptophan ratio) associated (p < 0.001) with poorer cognitive performance and greater likelihood of impairment. PASC, independent of disability associated with abnormal kynurenine/tryptophan (p < 0.03). INTERPRETATION: The kynurenine pathway relates to post-acute COVID-19 objective cognitive impairment and PASC, thereby enabling biomarker and therapeutic possibilities.

Association Between Tryptophan Metabolites, Physical Performance, and Frailty in Older Persons.

Frailty is defined as a syndrome of physiological decline in late life, characterized by marked vulnerability to adverse health outcomes. A robust biomarker for frailty is still lacking. Tryptophan (TRP) metabolism through the kynurenine pathway (KP) plays essential roles in aging, the musculoskeletal system, and physical performance. In this study, we quantified 7 KP metabolites, including kynurenine (KYN), kynurenine acid (KYNA), quinolinic acid (QUIN), picolinic acid (PIC), 3-hydroxykynurenine (3-HK), 3-hydroxyanthranilic acid (3-HAA), and anthranilic acid (AA) using ultra-high-performance liquid chromatography and gas chromatography-mass spectrometry in the serum of 85 participants (median age 75; 65% female; 28 non-frail, 29 pre-frail, and 28 frail) at the Nepean Osteoporosis and Frailty (NOF) Study. We looked at the association between TRP metabolites and physical performance, sarcopenia, and frailty. After adjusting for age and sex, our results showed that KYN and KYN/TRP were associated with higher interleukin (IL)-6 levels (r = .324 and r = .390, respectively). KYNA and its ratios to other products (mainly KYNA/KYN, KYNA/QUIN, and KYNA/PIC) were associated with a lower likelihood of frailty by Fried's criteria (OR 0.93 [0.88, 0.98], P = .009) and Rockwood index (r = -.241, P = .028) as well as a lower likelihood of sarcopenia (OR 0.88 [0.78, 1.00], P = .049). QUIN and QUIN/KYN showed an association with increased IL-6 (r = .293 and .204 respectively), higher likelihood of frailty (OR 1.02 [1.00, 1.04], P = .029 and OR 6.43 [2.23, 18.51], P = .001 respectively) and lower physical function (r = -.205 and r = -.292). In conclusion, different TRP metabolites have various associations with physical performance, frailty, and sarcopenia. Defining the underlying mechanisms may permit the development and validation of new biomarkers and therapeutics for frailty and musculoskeletal conditions targeting specific metabolites of the TRP catabolic pathway.

Anti-inflammatory activities of Coleus forsteri (formerly Plectranthus forsteri) extracts on human macrophages and chemical characterization.

Introduction: Formerly named Plectranthus forsteri, Coleus forsteri (Benth.) A.J.Paton, 2019 is a Lamiaceae traditionally used to treat flu-like symptoms and shock-related ecchymosis, especially in the Pacific region. Few studies investigated chemical composition and anti-inflammatory potential of this plant. Method: Herein, we investigated anti-inflammatory potential of C. forsteri ethanolic (ePE) and cyclohexane (cPE) plant extract on LPS-induced human macrophages models and quantified cytokines and quinolinic acid (QUIN) as inflammatory markers. Results: Our results show that extract of ePE and cPE significantly inhibit inflammatory cytokine IL-6 and TNF-α induced by LPS on PMA-derived THP-1 macrophages. QUIN production is also diminished under ePE and cPE treatment in activated human monocyte-derived macrophages (MDMs). Seven abietane diterpenes were characterized from C. forsteri cPE including coleon U (1), coleon U-quinone (2), 8α,9α-epoxycoleon U-quinone (3), horminone or 7α-hydroxyroyleanone (4), 6ß,7α-dihydroxyroyleanone (5), 7α-acetoxy-6ß-hydroxyroyleanone (6) and 7α-formyloxy-6ß-hydroxyroyleanone (7). Discussion: We discussed potential contributions of these molecules from C. forsteri extracts for their anti-inflammatory activities.

Effects of Tryptophan Supplementation and Exercise on the Fate of Kynurenine Metabolites in Mice and Humans.

The kynurenine pathway of tryptophan (TRP) degradation (KP) generates metabolites with effects on metabolism, immunity, and mental health. Endurance exercise training can change KP metabolites by changing the levels of KP enzymes in skeletal muscle. This leads to a metabolite pattern that favors energy expenditure and an anti-inflammatory immune cell profile and reduces neurotoxic metabolites. Here, we aimed to understand if TRP supplementation in untrained vs. trained subjects affects KP metabolite levels and biological effects. Our data show that chronic TRP supplementation in mice increases all KP metabolites in circulation, and that exercise reduces the neurotoxic branch of the pathway. However, in addition to increasing wheel running, we did not observe other effects of TRP supplementation on training adaptations, energy metabolism or behavior in mice. A similar increase in KP metabolites was seen in trained vs. untrained human volunteers that took a TRP drink while performing a bout of aerobic exercise. With this acute TRP administration, TRP and KYN were higher in the trained vs. the untrained group. Considering the many biological effects of the KP, which can lead to beneficial or deleterious effects to health, our data encourage future studies of the crosstalk between TRP supplementation and physical exercise.

Papaverine, a Phosphodiesterase 10A Inhibitor, Ameliorates Quinolinic Acid-Induced Synaptotoxicity in Human Cortical Neurons.

Phosphodiesterase-10A (PDE10A) hydrolyse the secondary messengers cGMP and cAMP, two molecules playing important roles in neurodevelopment and brain functions. PDE10A is associated to progression of neurodegenerative diseases like Alzheimer's, Parkinson's, Huntington's diseases, and a critical role in cognitive functions. The present study was undertaken to determine the possible neuroprotective effects and the associated mechanism of papaverine (PAP), a PDE10A isoenzyme inhibitor, against quinolinic acid (QUIN)-induced excitotoxicity using human primary cortical neurons. Cytotoxicity potential of PAP was analysed using MTS assay. Reactive oxygen species (ROS) and mitochondrial membrane potential were measured by DCF-DA and JC10 staining, respectively. Caspase 3/7 and cAMP levels were measured using ELISA kits. Effect of PAP on the CREB, BNDF and synaptic proteins such as SAP-97, synaptophysin, synapsin-I, and PSD-95 expression was analysed by Western blot. Pre-treatment with PAP increased intracellular cAMP and nicotinamide adenine dinucleotide (NAD+) levels, restored mitochondrial membrane potential (ΔΨm), and decreased ROS and caspase 3/7 content in QUIN exposed neurons. PAP up-regulated CREB and BDNF, and synaptic protein expression. In summary, these data indicate that PDE10A is involved in QUIN-mediated synaptotoxicity and its inhibition elicit neuroprotection by reducing the oxidative stress and protecting synaptic proteins via up-regulation of cAMP signalling cascade.

Chemical reprogramming enhances homology-directed genome editing in zebrafish embryos.

Precise genome editing is limited by the inefficiency of homology-directed repair (HDR) compared to the non-homologous end-joining (NHEJ) of double strand breaks (DSBs). The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system generates precise, locus-specific DSBs that can serve as substrates for HDR. We developed an in vivo visual reporter assay to quantify HDR-mediated events at single-cell resolution in zebrafish and used this system to identify small-molecule modulators that shift the DNA repair equilibrium in favor of HDR. By further optimizing the reaction environment and repair template, we achieved dramatic enhancement of HDR-mediated repair efficiency in zebrafish. Accordingly, under optimized conditions, inhibition of NHEJ with NU7441 enhanced HDR-mediated repair up to 13.4-fold. Importantly, we demonstrate that the increase in somatic HDR events correlates directly with germline transmission, permitting the efficient recovery of large seamlessly integrated DNA fragments in zebrafish.

Aurora kinase B regulates axonal outgrowth and regeneration in the spinal motor neurons of developing zebrafish.

Aurora kinase B (AurkB) is a serine/threonine protein kinase with a well-characterised role in orchestrating cell division and cytokinesis, and is prominently expressed in healthy proliferating and cancerous cells. However, the role of AurkB in differentiated and non-dividing cells has not been extensively explored. Previously, we have described a significant upregulation of AurkB expression in cultured cortical neurons following an experimental axonal transection. This is somewhat surprising, as AurkB expression is generally associated only with dividing cells Frangini et al. (Mol Cell 51:647-661, 2013); Hegarat et al. (J Cell Biol 195:1103-1113, 2011); Lu et al. (J Biol Chem 283:31785-31790, 2008); Trakala et al. (Cell Cycle 12:1030-1041, 2014). Herein, we present the first description of a role for AurkB in terminally differentiated neurons. AurkB was prominently expressed within post-mitotic neurons of the zebrafish brain and spinal cord. The expression of AurkB varied during the development of the zebrafish spinal motor neurons. Utilising pharmacological and genetic manipulation to impair AurkB activity resulted in truncation and aberrant motor axon morphology, while overexpression of AurkB resulted in extended axonal outgrowth. Further pharmacological inhibition of AurkB activity in regenerating axons delayed their recovery following UV laser-mediated injury. Collectively, these results suggest a hitherto unreported role of AurkB in regulating neuronal development and axonal outgrowth.

S100A8/A9 and S100A9 reduce acute lung injury.

S100A8 and S100A9 are myeloid cell-derived proteins that are elevated in several types of inflammatory lung disorders. Pro- and anti-inflammatory properties are reported and these proteins are proposed to activate TLR4. S100A8 and S100A9 can function separately, likely through distinct receptors but a systematic comparison of their effects in vivo are limited. Here we assess inflammation in murine lung following S100A9 and S100A8/A9 inhalation. Unlike S100A8, S100A9 promoted mild neutrophil and lymphocyte influx, possibly mediated in part, by increased mast cell degranulation and selective upregulation of some chemokine genes, particularly CXCL-10. S100 proteins did not significantly induce proinflammatory mediators including TNF-α, interleukin-1ß (IL-1ß), IL-6 or serum amyloid A3 (SAA3). In contrast to S100A8, neither preparation induced S100A8 or IL-10 mRNA/protein in airway epithelial cells, or in tracheal epithelial cells in vitro. Like S100A8, S100A9 and S100A8/A9 reduced neutrophil influx in acute lung injury provoked by lipopolysaccharide (LPS) challenge but were somewhat less inhibitory, possibly because of differential effects on expression of some chemokines, IL-1ß, SAA3 and IL-10. Novel common pathways including increased induction of an NAD+-dependent protein deacetylase sirtuin-1 that may reduce NF-κB signalling, and increased STAT3 activation may reduce LPS activation. Results suggest a role for these proteins in normal homeostasis and protective mechanisms in the lung.

A Tol2 Gateway-Compatible Toolbox for the Study of the Nervous System and Neurodegenerative Disease.

Currently there is a lack in fundamental understanding of disease progression of most neurodegenerative diseases, and, therefore, treatments and preventative measures are limited. Consequently, there is a great need for adaptable, yet robust model systems to both investigate elementary disease mechanisms and discover effective therapeutics. We have generated a Tol2 Gateway-compatible toolbox to study neurodegenerative disorders in zebrafish, which includes promoters for astrocytes, microglia and motor neurons, multiple fluorophores, and compatibility for the introduction of genes of interest or disease-linked genes. This toolbox will advance the rapid and flexible generation of zebrafish models to discover the biology of the nervous system and the disease processes that lead to neurodegeneration.

Exhaled breath markers of alveolar macrophage activity in sarcoidosis.

OBJECTIVE: Granuloma formation in sarcoidosis is dependent upon the interaction between alveolar macrophages (AMs) and a CD4+-driven TH1 response. This study aimed to measure TNF-α and calcium ion concentrations as markers of AM activity, in addition to total protein as a non-specific inflammatory marker in the exhaled breath condensate (EBC) of patients with sarcoidosis as well as control subjects. METHODS: EBC was collected from 17 sarcoidosis patients and 23 healthy volunteers. Protein was measured by the bicinchoninic acid assay, TNF-α concentration was measured by ELISA and Ca(2+) concentration was measured by inductively coupled plasma-mass spectrometry. Conductivity of EBC was assessed using a conductivity probe. RESULTS: Total protein concentration was significantly elevated in EBC from patients with sarcoidosis compared to control subjects (19.51 ± 4.52 vs. 10.60 ± 1.31 µg/ml, p = 0.020), as was TNF-α (3.37 ± 0.38 vs. 2.59 ± 0.40 pg/ml, p = 0.037) and conductivity (66.68 ± 16.73 vs. 36.85 ± 3.070 µS/cm, p = 0.044). EBC Ca(2+) concentration was significantly higher in healthy controls compared to patients with sarcoidosis (116.50 ± 12.19 vs. 73.88 ± 13.35 µmol/l, p = 0.018), although this was in the context of normal serum Ca(2+) in the sarcoidosis cohort. CONCLUSIONS: Total protein and TNF-α concentrations were elevated in EBC from patients with sarcoidosis and could indicate disease activity. The reduction in EBC Ca(2+) concentrations could represent granulomatous activity in the lung.

Clinical outcomes of xeno-free expansion and transplantation of autologous ocular surface epithelial stem cells via contact lens delivery: a prospective case series.

INTRODUCTION: Depletion of limbal stem cells leads to a debilitating condition known as limbal stem cell deficiency, characterised by impaired corneal wound healing and poor vision. The aim of this study was to determine whether delivering progenitor cells on a contact lens is a viable and effective alternative to current transplantation techniques, which are complicated by biological and xenogeneic materials. METHODS: Sixteen eyes of 16 patients who had total (n=14) and partial (n=2) limbal stem cell deficiency (chemical burns, five eyes; iatrogenic causes, four eyes; aniridia, three eyes; trachoma-induced, two eyes; contact lens over-wear, one eye; and cicatrising conjunctivitis, one eye) and who had failed prior therapy were recruited prospectively into the study. Autologous limbal (n=7) or conjunctival epithelial (n=9) biopsies were harvested from patients and placed on the concave surface of silicone hydrogel contact lenses. Cells were expanded in culture with autologous serum and transplanted onto the ocular surface. RESULTS: Restoration of a transparent avascular and clinically stable corneal epithelium was attained in 10 of 16 eyes (63%) at a median follow-up time of 2.5 years (range of 0.8 to 5.8 years). Although minor complications occurred in two eyes of two patients because of contact lens insertion or removal, these were not associated with long-term sequelae. CONCLUSIONS: This is the first and largest study to evaluate the mid-term outcomes of autologous limbal/conjunctival stem cell transplantation via a US Food and Drug Administration-approved contact lens, demonstrating that delivery of ocular progenitor cells via this procedure offers a viable, effective, and xeno-free alternative to current transplantation methodologies. TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry ACTRN012607000211460. Registered 17 April 2007.

Vitronectin: a migration and wound healing factor for human corneal epithelial cells.

PURPOSE: The stratified squamous epithelial covering of the cornea provides one of the first physical and immunological lines of defense. A breach in its integrity results in a wound healing response that resolves quickly. However, under certain conditions-for example, when migrating cells are unable to adhere to the basement membrane-healing may be delayed. The aim of this study was to determine whether vitronectin (VN) promotes corneal epithelial wound healing. METHODS: Primary human corneolimbal epithelial cells and the human corneal epithelial cell (HCEC) line were cultured and monitored to determine the rate of epithelial recovery after injury. Human corneas were placed in organ culture and the epithelium debrided. Therapeutic contact lenses (CLs) soaked in saline or coated in a solution of recombinant human VN were applied and the epithelium assessed histologically after 7 days. RESULTS: Vitronectin (5 µg/mL) significantly enhanced the wound closure rate in cultured HCECs as well as in primary human corneal epithelial cells. Wound recovery was significantly blocked with a cyclic arginine-glycine-aspartate (RGD) peptide (10 µg/mL), a neutralizing antibody to VN (25 µg/mL) as well as after transiently silencing ß5-integrin. Wound closure was not related to the effect of VN on cell proliferation. Finally, incubating epithelial-debrided human corneas with CLs loaded in VN resulted in effective re-epithelialization of the injured surface. CONCLUSIONS: Vitronectin is a key extracellular matrix protein that expedites corneal epithelial wound recovery in vitro and ex vivo. Delivery of VN on therapeutic CLs may be an effective and efficient way to treat patients with persistent epithelial defects of the ocular surface.

S100A8 induces IL-10 and protects against acute lung injury.

S100A8 is considered proinflammatory by activating TLR4 and/or the receptor for advanced glycation end products. The aim was to investigate inflammatory effects of S100A8 in murine lung. S100A8 was administered to BALB/c mice by nasal inhalation and genes induced over a time-course assessed. LPS was introduced intranasally either alone or 2 h after pretreatment of mice with intranasal application of S100A8 or dexamethasone. A Cys(42)-Ala(42) mutant S100A8 mutant was used to assess whether S100A8's effects were via pathways that were dependent on reactive oxygen species. S100A8 induced IL-10 mRNA, and expression was apparent only in airway epithelial cells. Importantly, it suppressed acute lung injury provoked by LPS inhalation by suppressing mast-cell activation and induction of mediators orchestrating leukocyte recruitment, possibly by reducing NF-κB activation via an IκBα/Akt pathway and by downmodulating pathways generating oxidative stress. The Cys(42)-Ala(42) S100A8 mutant did not induce IL-10 and was less immunosuppressive, indicating modulation by scavenging oxidants. S100A8 inhibition of LPS-mediated injury was as potent, and outcomes were remarkably similar to immunosuppression by dexamethasone. We challenge the notion that S100A8 is an agonist for TLR4 or the receptor for advanced glycation end products. S100A8 induced IL-10 in vivo and initiates a feedback loop that attenuates acute lung injury.

Detecting human papillomavirus in ocular surface diseases.

PURPOSE: Human papillomavirus (HPV) infection has been implicated as a possible inducing factor for benign and neoplastic ocular surface diseases such as pterygia and ocular-surface squamous neoplasia (OSSN). However, the wide range in HPV prevalence previously reported for both diseases adds controversy to, and highlights the limitations of, this field. The aim of this study was to determine the prevalence of HPV in pterygia and OSSN and to devise a standardized approach for detecting viral DNA in ocular tissue samples. METHODS: DNA was extracted from a variety of specimens (n = 160), including formalin-fixed paraffin-embedded tissue shavings, fresh tissue, and cultured cells. Nested PCR for HPV with consensus and subtype-specific primers was used to detect viral DNA. Confirmatory assays, including molecular sequencing, histology, and immunohistochemistry for HPV E6 protein and p16 were also performed. RESULTS: HPV was not detected in pterygia or normal conjunctiva. However, 6.5% (3/46) of OSSN samples were HPV-positive by PCR, sequencing, and immunohistochemistry. Positive cases were all squamous cell carcinoma of the conjunctiva (SCCC), the most severe form of OSSN, representing 12.5% (3/24) of SCCCs in our cohort. HPV-16 was the genotype identified in each case and this correlated with the presence of koilocytes and intense immunoreactivity for p16. Our study found no association between pterygia and OSSN with other oncogenic viruses, such as EBV or CMV, as they were just as prevalent in normal conjunctiva. CONCLUSIONS: The low prevalence of HPV-16 in ocular surface disease suggests infection is not a cause but a cofactor in disease development.

Exhaled breath condensate in pulmonary arterial hypertension.

Over the last decade, several new agents have been developed for the treatment of pulmonary arterial hypertension (PAH), and blood biomarkers have been developed which aim to monitor such treatment, and which correlate well with physiological parameters, symptoms and mortality. However, little is known regarding biomarkers collected using non-invasive methods such as exhaled breath condensate (EBC). EBC biomarkers show potential as a rapid, repeatable and easy method of sampling the pulmonary vasculature in severely ill patients. The current study aimed to investigate EBC biomarkers in patients with PAH of different aetiologies. We studied 89 patients in four groups: pulmonary arterial hypertension (PAH, n = 30), PAH associated with COPD (COPD/PAH, n = 14), COPD but no PAH (n = 16) and healthy controls (n = 29). Levels of the following EBC markers were measured: amino-terminal pro-brain natriuretic peptide (NT-proBNP), endothelin-1 (ET-1), 6-keto prostaglandin (PG)F(1α), hydrogen peroxide (H(2)O(2)), total oxides of nitrogen (NO(x)), total protein and pH. ET-1 and NT-proBNP were measured in plasma concurrently. Data were analysed with ANOVA or Kruskal Wallis tests where appropriate. Correlations were performed using Pearson's correlation coefficient. NT-proBNP was detectable in EBC and was highest in the PAH group, significantly higher than the COPD/PAH group (194.1 ± 23.3 versus 80.8 ± 22.2 fmol ml(-1), p < 0.05). EBC ET-1 was significantly higher in subjects with PAH (1.53 ± 0.32 fmol ml(-1)) compared to those with COPD/PAH (0.25 ± 0.03 fmol ml(-1), p < 0.05) and controls (0.66 ± 0.18 fmol ml(-1), p < 0.05). 6-keto PGF(1α) was low in the PAH group, significantly lower than the COPD/PAH group (4027 ± 445 versus 8381 ± 1024 pg ml(-1), p < 0.01). EBC biomarkers are measurable in PAH. EBC ET-1 was raised in PAH compared with controls and patients with PAH secondary to COPD, whereas 6-keto PGF(1α) was low. EBC biomarkers may be useful in detection and monitoring of PAH.

Exhaled breath condensate (EBC) biomarkers in pulmonary fibrosis.

The diffuse parenchymal lung diseases (DPLDs) are a group of clinicopathological entities which have recently undergone reclassification. The commonest type of idiopathic DPLD is interstitial pulmonary fibrosis (PF), which is histologically characterized by usual interstitial pneumonia (UIP), with inflammatory changes in the alveoli and subsequent collagen deposition. A similar type of inflammatory change can also be seen with connective tissue disorders. Many mediators are involved, but it is difficult to study these in a non-invasive manner in patients. The aim of the study detailed in this paper was to investigate inflammatory and oxidative stress biomarkers in PF and correlate these with lung function. 20 PF patients and 20 controls participated in the study. Exhaled breath condensate (EBC) was collected over 10 min using a refrigerated condenser, after fractional exhaled nitric oxide (FeNO) and carbon monoxide (eCO) measurement. EBC total nitrogen oxides (NOx), hydrogen peroxide (H(2)O(2)), 8-isoprostane (8-iso), 3-nitrotyrosine (3-NT), pH and total protein were measured. EBC biomarkers were significantly raised in PF compared with controls: EBC 3-NT (2.5 (0.7-8.9) versus 0.3 (0.1-1.1) ng ml(-1), p = 0.02); pH (7.6 ± 0.3 versus 7.4 ± 0.2, p = 0.004); 8-isoprostane (0.2 (0.1-0.4) versus 0.08 (0.04-0.2) ng ml(-1), p = 0.04) and total protein (24.7 ± 21.1 versus 10.7 ± 7.0 µg ml(-1), p = 0.008). FeNO and eCO were also increased (8.6 (7.1-10.4) versus 6.6 (5.6-7.8) ppb, p = 0.04, and 4.5 ± 1.7 versus 2.7 ± 0.7 ppm, p = 0.001, respectively), but no significant differences were found for NOx or H(2)O(2). In conclusion, inflammatory and oxidative stress biomarkers are raised in patients with PF compared with controls. EBC may be useful for detecting and monitoring lung inflammation in PF.

Vitronectin: a matrix support factor for human limbal epithelial progenitor cells.

PURPOSE: The authors recently developed a therapeutic technique for patients with limbal stem cell deficiency by harvesting ocular surface stem cells (SCs), expanding them on therapeutic contact lenses (CLs), and applying them to diseased corneas. The present study determined the proteins that bind to CLs and whether such factors, along with transplanted cells, are critical determinants for corneal rehabilitation using this method. METHODS: Therapeutic CLs were exposed to human serum, and adherent proteins were analyzed by proteomics. The distribution of vitronectin (VN) on the ocular surface was determined with specific antibodies. Cadaveric human corneas were chemically wounded, and cell transfer by CLs was assessed in organ culture. RESULTS: VN was identified as a serum factor that binds and desorbs from CLs. VN localized to the limbal and basement membranes (BM) of other SC-harboring organs. Clonogenic assays demonstrated higher colony-forming efficiency on VN compared with uncoated surfaces. Cell transfer from CLs was achieved through in vitro models and was abrogated by RGD peptides and inhibitory antibodies to VN and its receptor. CONCLUSIONS: Identification of VN within the limbal BM, its effect on limbal SC activity, and the discovery of this factor on serum-exposed CL polymers implies a role in supporting progenitor cells and facilitating corneal regeneration.