HGT in the human and skin commensal Malassezia: A bacterially derived flavohemoglobin is required for NO resistance and host interaction.

The skin of humans and animals is colonized by commensal and pathogenic fungi and bacteria that share this ecological niche and have established microbial interactions. Malassezia are the most abundant fungal skin inhabitant of warm-blooded animals and have been implicated in skin diseases and systemic disorders, including Crohn's disease and pancreatic cancer. Flavohemoglobin is a key enzyme involved in microbial nitrosative stress resistance and nitric oxide degradation. Comparative genomics and phylogenetic analyses within the Malassezia genus revealed that flavohemoglobin-encoding genes were acquired through independent horizontal gene transfer events from different donor bacteria that are part of the mammalian microbiome. Through targeted gene deletion and functional complementation in Malassezia sympodialis, we demonstrated that bacterially derived flavohemoglobins are cytoplasmic proteins required for nitric oxide detoxification and nitrosative stress resistance under aerobic conditions. RNA-sequencing analysis revealed that endogenous accumulation of nitric oxide resulted in up-regulation of genes involved in stress response and down-regulation of the MalaS7 allergen-encoding genes. Solution of the high-resolution X-ray crystal structure of Malassezia flavohemoglobin revealed features conserved with both bacterial and fungal flavohemoglobins. In vivo pathogenesis is independent of Malassezia flavohemoglobin. Lastly, we identified an additional 30 genus- and species-specific horizontal gene transfer candidates that might have contributed to the evolution of this genus as the most common inhabitants of animal skin.

Contact Lens Prescribing Trends for Keratoconus at an Academic Medical Center: Increased Utilization of Scleral Lenses for Severe Disease.

OBJECTIVES: To describe contact lens prescription trends for patients with keratoconus. METHODS: Demographics, corneal curvature, visual acuity, and type of contact lens prescribed were reviewed for all patients with keratoconus evaluated in the contact lens service in 2010 and 2020. RESULTS: There were 292 patients in 2010 and 217 in 2020. In 2010, 69% were using corneal gas-permeable lenses (GP), 16% soft toric, 13% hybrid, and 2% soft sphere with no scleral lens (SL). In 2020, 60% were using corneal GP, 22% SL, 12% soft toric, 5% hybrid, and 1% soft sphere. Mean log of minimum angle of resolution visual acuities with manifest refraction were (represented in mean [SD, range]) 0.42 (0.33, 0-1.3) in 2010 and 0.35 (0.33, 0-1.6) in 2020 improving to 0.19 (0.18, 0-1.3; P≤0.01) and 0.13 (0.14, 0-0.60; P<0.01) with contact lenses. Mean keratometry measurement in patients using SLs was 53.0 diopters (D) (9.9, 42.1-84.5), which was steeper than 46.6 D in patient's using all other lens types in 2010 and 2020 (3.9, 40.9-57.9; P<0.01). CONCLUSIONS: Scleral lens prescription increased during the past decade; however, corneal GP lenses remain the most frequent lens prescribed for patients with keratoconus in this cohort. Despite being prescribed for patients with the most advanced disease, SLs provide good visual acuity.

Pannexin 1 channels control the hemodynamic response to hypoxia by regulating O2-sensitive extracellular ATP in blood.

Pannexin 1 (Panx1) channels export ATP and may contribute to increased concentration of the vasodilator ATP in plasma during hypoxia in vivo. We hypothesized that Panx1 channels and associated ATP export contribute to hypoxic vasodilation, a mechanism that facilitates the matching of oxygen delivery to metabolic demand of tissue. Male and female mice devoid of Panx1 (Panx1-/-) and wild-type controls (WT) were anesthetized, mechanically ventilated, and instrumented with a carotid artery catheter or femoral artery flow transducer for hemodynamic and plasma ATP monitoring during inhalation of 21% (normoxia) or 10% oxygen (hypoxia). ATP export from WT vs. Panx1-/-erythrocytes (RBC) was determined ex vivo via tonometer experimentation across progressive deoxygenation. Mean arterial pressure (MAP) was similar in Panx1-/- (n = 6) and WT (n = 6) mice in normoxia, but the decrease in MAP in hypoxia seen in WT was attenuated in Panx1-/- mice (-16 ± 9% vs. -2 ± 8%; P < 0.05). Hindlimb blood flow (HBF) was significantly lower in Panx1-/- (n = 6) vs. WT (n = 6) basally, and increased in WT but not Panx1-/- mice during hypoxia (8 ± 6% vs. -10 ± 13%; P < 0.05). Estimation of hindlimb vascular conductance using data from the MAP and HBF experiments showed an average response of 28% for WT vs. -9% for Panx1-/- mice. Mean venous plasma ATP during hypoxia was 57% lower in Panx1-/- (n = 6) vs. WT mice (n = 6; P < 0.05). Mean hypoxia-induced ATP export from RBCs from Panx1-/- mice (n = 8) was 82% lower than that from WT (n = 8; P < 0.05). Panx1 channels participate in hemodynamic responses consistent with hypoxic vasodilation by regulating hypoxia-sensitive extracellular ATP levels in blood.NEW & NOTEWORTHY Export of vasodilator ATP from red blood cells requires pannexin 1. Blood plasma ATP elevations in response to hypoxia in mice require pannexin 1. Hemodynamic responses to hypoxia are accompanied by increased plasma ATP in mice in vivo and require pannexin 1.

Comparison of International Ovarian Tumor Analysis Simple Rules to Society of Radiologists in Ultrasound Guidelines for Detection of Malignancy in Adnexal Cysts.

OBJECTIVE. The purpose of this study was to evaluate the International Ovarian Tumor Analysis (IOTA) simple rules and the Society of Radiologists in Ultrasound (SRU) guidelines for detecting ovarian malignancy in a general population of women presenting to radiology departments with adnexal cystic lesions. MATERIALS AND METHODS. A retrospective multicenter study of ultrasound-detected adnexal cystic lesions with appropriate follow-up was conducted. Lesions were classified into benign, indeterminate, or malignant categories according to criteria based on the IOTA simple rules and the SRU guidelines. The prevalence of nonneoplastic cysts, neoplasms, and malignant tumors was calculated. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and accuracy were calculated, and ROC analysis for the diagnosis of malignancy was performed. RESULTS. A total of 697 women with 764 cystic lesions were included; 85.2% (651/764) of the lesions were nonneoplastic, 12.2% (93/764) were benign neoplasms, and 2.6% (20/764) were malignant neoplasms. Nearly all malignancies were classified into indeterminate and malignant categories. The prevalence of malignancy in the indeterminate category was 4.8% (7/145) (SRU) to 10.7% (7/65) (IOTA) and in the malignant category was 18.1% (13/72) (SRU) to 34.3% (12/35) (IOTA). Only one malignancy was misclassified as benign by the IOTA simple rules. The sensitivity of the IOTA simple rules for malignancy was 90.0%; specificity, 96.5%; PPV, 29.0%; NPV, 99.8%; and accuracy, 96.4%. The corresponding values for the SRU guidelines were 100%, 89.6%, 14.9%, 100%, and 89.8%. In ROC analysis, the IOTA simple rules were slightly more accurate than the SRU guidelines (AUC, 0.9805 versus 0.9713; p = 0.0003). CONCLUSION. Both imaging characterization methods were sensitive for identifying ovarian malignancies, but the PPV was low among women presenting to radiology departments, and the indeterminate classification harbored one-third of the total malignancies. Exploration of varied clinical settings and inclusion of secondary tests may help to refine these systems.

Prevalence of Ocular Surface Disease and Corneal Irregularity and Outcomes in Patients Using Therapeutic Scleral Lenses at a Tertiary Care Center.

OBJECTIVES: To describe indications for scleral contact lens (ScCL) evaluation, previous treatments, and outcomes of patients prescribed ScCL at a tertiary referral center. METHODS: This retrospective study reviewed 133 patients evaluated for ScCL between January 1, 2010, and December 31, 2015, at the University of Illinois at Chicago (UIC) Contact Lens Service. Patient demographics, ocular history, indications for evaluation, previous treatments, presence of punctate epithelial erosions, number of lenses ordered, follow-up visits, best-corrected visual acuity before ScCL, and visual acuity with ScCL were evaluated. Patients were categorized based on primary indication for ScCL evaluation as ocular surface disease (OSD) or corneal irregularity (CI). The primary outcome was visual acuity. RESULTS: Visual acuity improved from logarithm of the minimal angle of resolution 0.3±0.4 at presentation to 0.1±0.2 with ScCL in all eyes (n=223, P=0.0001), and from 0.3±0.5 to 0.1±0.2 (n=164, P=0.001) in the OSD group and 0.5±0.4 to 0.2±0.2 (n=59, P=0.0001) in the CI group. Seventy percent of patients were evaluated for bilateral ScCL evaluation. Indication for ScCL evaluation was OSD in 71% (n=95) of patients, with 20% having a secondary diagnosis of CI. Corneal irregularity was the primary diagnosis in 29% of patients (n=38), with 50% having a secondary diagnosis of OSD. CONCLUSIONS: Ocular surface disease was common in this tertiary referral patient population and was a primary or secondary indication for ScCL evaluation in 85.7% (n=114) of patients evaluated for ScCL. Scleral contact lenses improved visual outcomes in patients with both primary diagnoses of CI and OSD.

Indeterminate Adnexal Cysts at US: Prevalence and Characteristics of Ovarian Cancer.

Purpose To assess the prevalence of indeterminate adnexal cysts in women presenting to academic medical centers for pelvic ultrasonography (US), determine the incidence of malignancy, and identify cyst and patient characteristics that are predictive of malignancy. Materials and Methods A multicenter study of US-detected adnexal cysts with appropriate follow-up (surgical pathologic examination, imaging and/or clinical examination) was conducted from January 2008 to June 2012. Indeterminate cysts were classified as category 1 (typical benign appearing cysts >5 cm) or category 2 (cysts with avascular solid components) on the basis of a combination of definitions in the existing literature. The incidence of neoplasms and malignant tumors was calculated. Patient and cyst characteristics associated with neoplasm and malignant tumors were evaluated with the χ2 test or Fisher exact test for categorical variables and the t test for continuous variables. A backward stepwise logistic regression model was performed for two outcomes: (a) the presence of any neoplasm (benign or malignant) and (b) the presence of a malignant tumor. Results There were 1637 women with an adnexal cyst at US; 391 (mean age = 41.8 years ± 13.5.1; range = 17-91 years) had an indeterminate adnexal cyst at US. The prevalence of indeterminate adnexal cysts was 23.9% (391 of 1637; 95% confidence interval [CI]: 0.22, 0.26). Three hundred three indeterminate cysts in 280 women (mean age = 42.9 years ± 14.1; range = 17-88 years) had adequate follow-up. The incidence of ovarian neoplasms (benign and malignant) was 24.8% (75 of 303 cysts; 95% CI: 0.20, 0.30), and the incidence of malignant tumors was 3.6% (11 of 303 cysts; 95% CI: 0.02, 0.06). The proportion of ovarian neoplasms differed between category 1 and category 2 cysts (17.5% [25 of 143 cysts; 95% CI: 0.12, 0.25] vs 31.3% [50 of 160 cysts; 95% CI: 0.24, 0.39], respectively; P = .001). The proportion of malignant tumors differed between categories 1 and 2 cysts (0% [0 of 143 cysts] vs 6.9% [11 of 160 cysts; 95% CI: 0.03, 0.12]; P < .001). The presence of an avascular nodular component was a significant predictor of malignancy at stepwise logistic regression analysis (odds ratio = 2.83; P ≤ .0001; 95% CI: 1.69, 4.70). Conclusion The presence of an avascular nodular component was the most significant predictor of the presence of malignancy in indeterminate adnexal cysts. The risk of malignancy is higher with category 2 cysts than with category 1 cysts. © RSNA, 2018.

Proceedings of the Food and Drug Administration's public workshop on new red blood cell product regulatory science 2016.

The US Food and Drug Administration (FDA) held a workshop on red blood cell (RBC) product regulatory science on October 6 and 7, 2016, at the Natcher Conference Center on the National Institutes of Health (NIH) Campus in Bethesda, Maryland. The workshop was supported by the National Heart, Lung, and Blood Institute, NIH; the Department of Defense; the Office of the Assistant Secretary for Health, Department of Health and Human Services; and the Center for Biologics Evaluation and Research, FDA. The workshop reviewed the status and scientific basis of the current regulatory framework and the available scientific tools to expand it to evaluate innovative and future RBC transfusion products. A full record of the proceedings is available on the FDA website (http://www.fda.gov/BiologicsBloodVaccines/NewsEvents/WorkshopsMeetingsConferences/ucm507890.htm). The contents of the summary are the authors' opinions and do not represent agency policy.

S-Nitrosylated fetal hemoglobin in neonatal human blood.

BACKGROUND: Nitric oxide (NO) and its derivatives play important roles in the cardiopulmonary transition upon birth and in other oxygen-sensitive developmental milestones. One mechanism for the coupling of oxygen sensing and signaling by NO species is via the formation of an S-nitrosothiol (SNO) moiety on hemoglobin (Hb, forming SNO-Hb) and its release from the red blood cell in hypoxia. Although SNO-Hb formed on adult-type Hb (HbA, forming SNO-HbA) has been documented in physiological and pathophysiological human states, the fetal variant, SNO-HbF, has thus far not been isolated or characterized in human blood. METHODS AND RESULTS: We developed a technique capable of separating Hbs A and F under conditions that preserve SNO. We then measured SNO-HbF in the blood of healthy and premature or otherwise ill neonates using the gold standard for SNO measurement, mercury-coupled photolysis-chemiluminescence. SNO-HbF levels were in the range of those previously reported for HbA in adults. We found that SNO-HbF was more abundant at earlier gestational age (<30 weeks), even when accounting for the absolute HbF level. CONCLUSIONS: The ability to monitor SNO-HbF could provide new insights into fetal development and the perinatal transition, and has potential as a biomarker relevant to the management of neonatal diseases.

Contact Lens Use in Patients With Boston Keratoprosthesis Type 1: Fitting, Management, and Complications.

The Boston type 1 keratoprosthesis (KPro) is the most commonly used artificial cornea worldwide. Long-term bandage contact lenses are the standard of care for patients with these devices. The goal of bandage contact lenses is to maintain hydration and to protect the corneal tissue that surrounds the anterior plate of the keratoprosthesis which is vulnerable to desiccation, epithelial breakdown, dellen formation, and corneal melt. Contact lenses can also improve comfort, correct refractive errors, and improve the cosmesis of patients with artificial corneas. However, the continuous use of contact lenses places these patients at risk for complications such as lens loss, lens deposits, chronic conjunctivitis, and infection. In addition, obtaining an adequate fit in a patient with a compromised ocular surface and history of multiple surgeries including glaucoma drainage devices can present a challenge. This review discusses the types of contact lenses used, special fitting considerations, and common complications in patients with previous KPro surgery.

Restoration of intracellular ATP production in banked red blood cells improves inducible ATP export and suppresses RBC-endothelial adhesion.

Transfusion of banked red blood cells (RBCs) has been associated with poor cardiovascular outcomes. Storage-induced alterations in RBC glycolytic flux, attenuated ATP export, and microvascular adhesion of transfused RBCs in vivo could contribute, but the underlying mechanisms have not been tested. We tested the novel hypothesis that improving deoxygenation-induced metabolic flux and the associated intracellular ATP generation in stored RBCs (sRBCs) results in an increased extracellular ATP export and suppresses microvascular adhesion of RBCs to endothelium in vivo following transfusion. We show deficient intracellular ATP production and ATP export by human sRBCs during deoxygenation (impairments ~42% and 49%, respectively). sRBC pretreatment with a solution containing glycolytic intermediate/purine/phosphate precursors (i.e., "PIPA") restored deoxygenation-induced intracellular ATP production and promoted extracellular ATP export (improvement ~120% and 50%, respectively). In a nude mouse model of transfusion, adhesion of human RBCs to the microvasculature in vivo was examined. Only 2% of fresh RBCs (fRBCs) transfused adhered to the vascular wall, compared with 16% of sRBCs transfused. PIPA pretreatment of sRBCs significantly reduced adhesion to just 5%. In hypoxia, adhesion of sRBCs transfused was significantly augmented (up to 21%), but not following transfusion of fRBCs or PIPA-treated sRBCs (3.5% or 6%). Enhancing the capacity for deoxygenation-induced glycolytic flux within sRBCs increases their ability to generate intracellular ATP, improves the inducible export of extracellular anti-adhesive ATP, and consequently suppresses adhesion of stored, transfused RBCs to the vascular wall in vivo.

Randomized study of washing 40- to 42-day-stored red blood cells.

BACKGROUND: Pretransfusion washing of red blood cells (RBCs) stored for a longer duration may have theoretical advantages but few data exist to support this practice. In many hospital settings, use of a point-of-care cell washer could conceivably be used to quickly wash allogeneic RBCs before transfusion. The purpose of this preliminary study was to compare a point-of-care device with a common blood bank device for washing longer-stored RBCs. STUDY DESIGN AND METHODS: Forty RBC units stored for 40 to 42 days were randomized to washing with the COBE 2991 device (Terumo BCT; FDA-cleared for washing stored RBCs) or the Cell Saver Elite (Haemonetics; FDA-cleared point-of-care device for processing and washing fresh autologous shed whole blood). Supernatant and unit RBCs from unwashed (baseline) and washed blood were assayed for potassium, lactate, intracellular ATP, percentage of RBC recovery, cell-free hemoglobin, RBC microparticles, and RBCs were examined for susceptibility to hemolysis by physical stress. RESULTS: Both devices recovered a high percentage of RBCs and efficiently removed extracelluar potassium. Washing with the Elite resulted in significant increases in cell-free Hb, percent hemolysis, and RBC microparticle production, whereas washing with the COBE 2991 did not (fold Δ = 2.1 vs. 1.0, 4.6 vs. 1.2, 2.0 vs. 1.1, respectively; p < 0.05). Hemolysis induced by physical stress was not altered by washing. CONCLUSION: Although point-of-care washing of longer-stored RBCs is appealing, these preliminary data suggest that transfusion of washed, longer-stored units could result in potentially greater exposure to plasma free Hb. More data are needed before this practice can be routinely recommended.

Toward Translational Impact of Low-Glucose Strategies on Red Blood Cell Storage Optimization.

Transfusion of stored red blood cells (RBCs) to patients is a critical component of human healthcare. Following purification from whole blood, RBCs are stored in one of many media known as additive solutions for up to 42 days. However, during the storage period, the RBCs undergo adverse chemical and physical changes that are often collectively known as the RBC storage lesion. Storage of RBCs in additive solutions modified to contain physiological levels of glucose, as opposed to hyperglycemic levels currently used in most cases, reduces certain markers of the storage lesion, although intermittent doses of glucose are required to maintain normoglycemic conditions. Here, we describe an electrically actuated valving system to dispense small volumes of glucose into 100 mL PVC storage bags containing packed RBCs from human donors. The RBCs were stored in a conventional additive solution (AS-1) or a normoglycemic version of AS-1 (AS-1N) and common markers of stored RBC health were measured at multiple time points throughout storage. The automated feeding device delivered precise and predictable volumes of concentrated glucose to maintain physiological glucose levels for up to 37 days. Hemolysis, lactate accumulation, and pH values of RBCs stored in AS-1N were statistically equivalent to values measured in AS-1, while significant reductions in osmotic fragility and intracellular sorbitol levels were measured in AS-1N. The reduction of osmotic fragility and oxidative stress markers in a closed system may lead to improved transfusion outcomes for an important procedure affecting millions of people each year.

Unearthing who and Y at Harewood Cemetery and inference of George Washington's Y-chromosomal haplotype.

An excavation conducted at Harewood Cemetery to identify the unmarked grave of Samuel Washington resulted in the discovery of burials presumably belonging to George Washington's paternal grandnephews and their mother, Lucy Payne. To confirm their identities this study examined Y-chromosomal, mitochondrial, and autosomal DNA from the burials and a living Washington descendant. The burial's Y-STR profile was compared to FamilyTreeDNA's database, which resulted in a one-step difference from the living descendant and an exact match to another Washington. A more complete Y-STR and Y-SNP profile from the descendant was inferred to be the Washington Y profile. Kinship comparisons performed in relation to the descendant, who is a 4th and 5th degree relative of the putative individuals, resulted in >37,000 overlapping autosomal SNPs and strong statistical support with likelihood ratios exceeding one billion. This study highlights the benefits of a multi-marker approach for kinship prediction and DNA-assisted identification of historical remains.

Automated measurement of blood flow velocity and direction and hemoglobin oxygen saturation in the rat lung using intravital microscopy.

Intravital microscopy of the pulmonary microcirculation in research animals is of great scientific interest for its utility in identifying regional changes in pulmonary microcirculatory blood flow. Although feasibility studies have been reported, the pulmonary window can be further refined into a practical tool for pharmaceutical research and drug development. We have established a method to visualize and quantify dynamic changes in three key features of lung function: microvascular red blood cell velocity, flow direction, and hemoglobin saturation. These physiological parameters were measured in an acute closed-chest pulmonary window, which allows real-time images to be captured by fluorescence and multispectral absorption microscopy; images were subsequently quantified using computerized analysis. We validated the model by quantifying changes in microcirculatory blood flow and hemoglobin saturation in two ways: 1) after changes in inspired oxygen content and 2) after pharmacological reduction of pulmonary blood flow via treatment with the ß1 adrenergic receptor blocker metoprolol. This robust and relatively simple system facilitates pulmonary intravital microscopy in laboratory rats for pharmacological and physiological research.

Proteomic analysis of the NOS2 interactome in human airway epithelial cells.

The cytokine-inducible isoform of nitric oxide synthase (NOS2) is constitutively expressed in human respiratory epithelia and is upregulated in inflammatory lung disease. Here, we sought to better define the protein interactions that may be important for NOS2 activity and stability, as well as to identify potential targets of NOS2-derived NO, in the respiratory epithelium. We overexpressed Flag-tagged, catalytically-inactive NOS2 in A549 cells and used mass spectrometry to qualitatively identify NOS2 co-immunoprecipitating proteins. Stable isotope labeling of amino acids in cell culture (SILAC) was used to quantify the coordinate effects of cytokine stimulation on NOS2-protein interactions. Multi-protein networks dominated the NOS2 interactome, and cytokine-inducible interactions with allosteric activators and with the ubiquitin-proteasome system were correlated with cytokine-dependent increases in NO metabolites and in NOS2 ubiquitination. The ubiquitin ligase scaffolding protein, FBXO45, was identified as a novel, direct NOS2 interactor. Similar to the SPRY domain-containing SOCS box (SPSB) proteins, FBXO45 requires Asn27 in the (23)DINNN(27) motif of NOS2 for its interaction. However, FBXO45 is unique from the SPSBs in that it recruits a distinct E3 ligase complex containing MYCBP2 and SKP1. Collectively, these findings demonstrate the general utility of interaction proteomics for defining new aspects of NOS2 physiology.

Contact lens surveillance cultures in Boston type 1 keratoprosthesis patients.

OBJECTIVES: To describe surveillance cultures of bandage contact lenses (BCL) in patients with a history of Boston type 1 keratoprosthesis (KPro) surgery. METHODS: An institutional review board-approved retrospective review of patients with a history of Boston type 1 KPro surgery and BCL cultures between July 2008 and June 2010. Data on demographics, preoperative diagnosis, topical corticosteroid and antibiotic use, duration of BCL wear, clinical diagnosis of microbial keratitis or endophthalmitis, and culture results were recorded. RESULTS: There were 14 patients (15 eyes) who underwent Boston type 1 KPro between July 2008 and June 2010 with BCL culture data. Ten eyes showed positive growth and one eye developed an infection. Thirty-four cultures were performed and 12 were positive. The most common organism cultured was coagulase-negative staphylococcus (CoNS). Positive cultures, colonization by non-CoNS pathogens, and infection occurred more frequently in patients with an ocular history of autoimmune disease or chemical burn. The growth of 15 or more CoNS colonies occurred only in patients not on vancomycin. An antibiotic was added to the regimen in response to positive cultures for three patients. CONCLUSIONS: Contact lens surveillance cultures may provide valuable information on the characteristics of microbial colonization, particularly in highlighting inflammatory disease and disuse of vancomycin as possible risk factors. Bandage contact lenses cultures also have the potential to be useful adjuncts in the clinical management of antibiotic therapy.

Transpulmonary flux of S-nitrosothiols and pulmonary vasodilation during nitric oxide inhalation: role of transport.

Inhaled nitric oxide (iNO) is used to treat pulmonary hypertension and is being investigated for prevention of bronchopulmonary dysplasia in neonates. Extrapulmonary effects of iNO are widely recognized, but the underlying chemistry and pharmacology are poorly understood. Growing evidence suggests that, in addition to acting via diffusion, NO can be converted into nitrosants capable of reacting with endogenous L-cysteine (L-Cys) in the alveolar lining fluid, forming S-nitrosothiol (SNO)-L-cysteine (CSNO). CSNO can then enter cells via the type L amino acid transporter (LAT). To determine the influence of LAT and supplemental L-Cys on the functional activity of iNO and transpulmonary movement of SNOs or other related species, we exposed C57Bl6 mice to nebulized L-Cys or D-cysteine (D-Cys) and/or LAT competitors. Isolated lungs were then perfused with physiologic buffer while effluent was collected to assay perfusate SNOs. Nebulized L-Cys, but not D-Cys, augmented the iNO-induced increase in circulating SNOs in the effluent without altering iNO-induced pulmonary vasodilation. Addition to the perfusate of either L-leucine (L-Leu) or 2-amino-2-norborane carboxylic acid, two distinct LAT competitors, inhibited appearance in the perfusate of SNOs in L-Cys-exposed lungs; a higher concentration of L-Leu significantly inhibited the iNO-induced pulmonary vasodilation as well as SNO accumulation. We conclude that iNO-induced pulmonary vasodilation and the transpulmonary movement of iNO-derived SNOs are mediated in part by formation of extracellular CSNO, uptake by alveolar epithelial LAT, and/or export by LAT from the pulmonary endothelium into the circulation. Therapies that exploit and optimize LAT-dependent SNO transport might improve the efficacy of and clinical outcomes with NO-based therapy by improving systemic SNO delivery.

Inhaled nitric oxide therapy increases blood nitrite, nitrate, and s-nitrosohemoglobin concentrations in infants with pulmonary hypertension.

OBJECTIVE: To measure the circulating concentrations of nitric oxide (NO) adducts with NO bioactivity after inhaled NO (iNO) therapy in infants with pulmonary hypertension. STUDY DESIGN: In this single center study, 5 sequential blood samples were collected from infants with pulmonary hypertension before, during, and after therapy with iNO (n = 17). Samples were collected from a control group of hospitalized infants without pulmonary hypertension (n = 16) and from healthy adults for comparison (n = 12). RESULTS: After beginning iNO (20 ppm) whole blood nitrite levels increased approximately two-fold within 2 hours (P<.01). Whole blood nitrate levels increased to 4-fold higher than baseline during treatment with 20 ppm iNO (P<.01). S-nitrosohemoglobin increased measurably after beginning iNO (P<.01), whereas iron nitrosyl hemoglobin and total hemoglobin-bound NO-species compounds did not change. CONCLUSION: Treatment of pulmonary hypertensive infants with iNO results in increases in levels of nitrite, nitrate, and S-nitrosohemoglobin in circulating blood. We speculate that these compounds may be carriers of NO bioactivity throughout the body and account for peripheral effects of iNO in the brain, heart, and other organs.

Impact of transfusion of autologous 7- versus 42-day-old AS-3 red blood cells on tissue oxygenation and the microcirculation in healthy volunteers.

BACKGROUND: Stored red blood cells (RBCs) accumulate biochemical and biophysical changes. Maximum storage duration is based on acceptable in vitro characteristics and 24-hour survival, but not RBC function. Relatively little is known about the impact of RBC storage duration on oxygenation and the microcirculation. STUDY DESIGN AND METHODS: Eight healthy subjects donated a double RBC apheresis, which were prestorage leukoreduced and processed in AS-3. Subjects were transfused 1 unit of RBCs at 7 and 42 days after blood collection. Measurements of percentage of tissue oxygenation in the thenar eminence muscle (StO2) and brain (SctO2) were recorded with Food and Drug Administration-cleared noninvasive devices. Sublingual microvascular blood flow (microcirculatory flow index [MFI]) was quantified before and after RBC transfusion using a video microscope. Raw electronic data for all measurements were analyzed by a blinded observer at a core laboratory. RESULTS: The only pre- versus posttransfusion change observed in measurements of SctO2, StO2, or MFI was a very small increase in SctO2, from 70.4 to 71.8 (means, p=0.032) at 7 days. There was no significant difference in the amount of pre-post change at 7 days versus 42 days for any of the measures. CONCLUSION: Transfusion of 1 unit of 42-day-stored RBCs to healthy subjects has no overt detrimental effect on tissue oxygenation or the microcirculation assessed by clinically available monitors.