Sex-dependent phosphorylation of Argonaute 2 reduces the mitochondrial translocation of miR-181c and induces cardioprotection in females.

Obesity-induced cardiac dysfunction is growing at an alarming rate, showing a dramatic increase in global prevalence. Mitochondrial translocation of miR-181c in cardiomyocytes results in excessive reactive oxygen species (ROS) production during obesity. ROS causes Sp1, a transcription factor for MICU1, to be degraded via post-translational modification. The subsequent decrease in MICU1 expression causes mitochondrial Ca2+ accumulation, ultimately leading to a propensity for heart failure. Herein, we hypothesized that phosphorylation of Argonaute 2 (AGO2) at Ser 387 (in human) or Ser 388 (in mouse) inhibits the translocation of miR-181c into the mitochondria by increasing the cytoplasmic stability of the RNA-induced silencing complex (RISC). Initially, estrogen offers cardioprotection in pre-menopausal females against the consequences of mitochondrial miR-181c upregulation by driving the phosphorylation of AGO2. Neonatal mouse ventricular myocytes (NMVM) treated with insulin showed an increase in pAGO2 levels and a decrease in mitochondrial miR-181c expression by increasing the binding affinity of AGO2-GW182 in the RISC. Thus, insulin treatment prevented excessive ROS production and mitochondrial Ca2+ accumulation. In human cardiomyocytes, we overexpressed miR-181c to mimic pathological conditions, such as obesity/diabetes. Treatment with estradiol (E2) for 48 h significantly lowered miR-181c entry into the mitochondria through increased pAGO2 levels. E2 treatment also normalized Sp1 degradation and MICU1 transcription that normally occurs in response to miR-181c overexpression. We then investigated these findings using an in vivo model, with age-matched male, female and ovariectomized (OVX) female mice. Consistent with the E2 treatment, we show that female hearts express higher levels of pAGO2 and thus, exhibit higher association of AGO2-GW182 in cytoplasmic RISC. This results in lower expression of mitochondrial miR-181c in female hearts compared to male or OVX groups. Further, female hearts had fewer consequences of mitochondrial miR-181c expression, such as lower Sp1 degradation and significantly decreased MICU1 transcriptional regulation. Taken together, this study highlights a potential therapeutic target for conditions such as obesity and diabetes, where miR-181c is upregulated. NEW AND NOTEWORTHY: In this study, we show that the phosphorylation of Argonaute 2 (AGO2) stabilizes the RNA-induced silencing complex in the cytoplasm, preventing miR-181c entry into the mitochondria. Furthermore, we demonstrate that treatment with estradiol can inhibit the translocation of miR-181c into the mitochondria by phosphorylating AGO2. This ultimately eliminates the downstream consequences of miR-181c overexpression by mitigating excessive reactive oxygen species production and calcium entry into the mitochondria.

Preoperative Liver Stiffness is Associated With Hospital Length of Stay After Cardiac Surgery.

OBJECTIVES: Risk assessment models for cardiac surgery do not account for the degrees of liver dysfunction. Ultrasound shear-wave elastography measures liver stiffness (LSM), a quantitative measurement related to fibrosis, congestion, and inflammation. The authors hypothesized that preoperative liver stiffness would be associated with hospital length of stay after cardiac surgery. DESIGN: Prospective observational study. SETTING: University hospital, single center. PARTICIPANTS: One hundred five adult patients undergoing nonemergent cardiac surgery. INTERVENTIONS: Preoperative liver stiffness measured by ultrasound elastography. MEASUREMENTS AND MAIN RESULTS: The associations were analyzed using linear mixed models, with adjustments for preoperative variables, duration of cardiopulmonary bypass, and type of surgery. Median liver stiffness was 6.4 kPa (range, 4.1-18.6 kPa). The median length of hospital stay was 6 days (range, 3-18 d). Each unit increase in liver stiffness, treated as a continuous variable, was associated with an increase of 0.32 ± 0.10 days in the hospital (p = 0.002). When treated as a categorical variable (<6 kPa, 6-9.4 kPa, and ≥9.5 kPa), LSM ≥9.5 kPa v LSM <6 kPa was associated strongly with an increase in hospital length of stay of 3.25 ± 0.87 days (p = 0.0003). CONCLUSIONS: A preoperative LSM ≥9.5 kPa was associated with a significantly longer postoperative hospital length of stay. This association appeared independent of preoperative comorbidities commonly associated with coronary disease. Preoperative liver stiffness is a novel risk metric that is associated with the postoperative hospital length of stay after cardiac surgery.

Mixing commonly used crystalloid solutions with red blood cells in five common additives does not negatively impact hemolysis, aggregometry, or deformability.

BACKGROUND: Literature is beginning to challenge the belief that it is unsafe to coinfuse red blood cells (RBCs) with solutions other than isotonic saline. We recently showed that additive-free RBCs tolerated coincubation with Plasma-Lyte or catecholamines dissolved in normal saline (NS), though 5% dextrose in water (D5W) promoted hemolysis. Herein, we evaluate the effect of coincubating crystalloids on additive-preserved RBC hemolysis, aggregation, and membrane deformability. STUDY DESIGN AND METHODS: RBCs were coincubated 5 minutes with plasma, NS, Plasma-Lyte, lactated Ringer's (LR) or D5W (1 mL PRBC +131.3 µL solution). Samples were then assessed for hemolysis (free hemoglobin), aggregation (critical shear stress [mPa]), and membrane deformability (elongation index [EI]). Significance (P ≤ .05) by t test or ANOVA with post-hoc Tukey-Kramer test. RESULTS: Additive-prepared RBCs coincubated with crystalloid instead of plasma demonstrated: (a) no increase in hemolysis as indicated by plasma free hemoglobin levels that is likely to be clinically relevant; (b) no increase, but in some cases a decrease, in aggregation as indicated by critical shear stress; and (c) in some combinations, a deterioration in deformability. When present, the deformability decrease was likely clinically insignificant in degree, and always returned to normal when the crystalloid was subsequently diluted out with plasma. CONCLUSION: Our data suggest that additive-prepared RBCs coincubated for 5 minutes with any of four common crystalloids demonstrate no clinically relevant increased lysis, increased aggregation, or decreased deformability.

Effect of incubation with crystalloid solutions or medications on packed red blood cells.

BACKGROUND: American Association of Blood Banks (AABB) guidelines suggest that packed red blood cells (PRBCs) be administered through a dedicated intravenous (IV) catheter. Literature supporting this broad-scope declaration are scarce. Obtaining additional IV access is painful, costly, and an infectious risk. We evaluated the effect of co-incubating PRBCs with crystalloids and medications on PRBC hemolysis, membrane deformability, and aggregation, as well as medication concentration. METHODS: PRBCs were co-incubated 5 minutes with plasma, normal saline (NS), 5% dextrose in water (D5W), Plasmalyte, epinephrine (epi), norepinephrine (norepi), dopamine (dopa), or Propofol (prop). Samples were then assessed for hemolysis (free hemoglobin, serum potassium), membrane deformability (elongation index [EI]), aggregation (smear, critical shear stress [mPa]) and drug concentration (High Performance Liquid Chromatography/Tandem Mass Spectrometry [LCMS-MS]). Significance (p ≤ 0.05) was determined by Wilcoxon-paired comparisons or Wilcoxon/Kruskall Willis with post-hoc Dunn's test. RESULTS: Compared to co-incubation with plasma: 1) co-incubation resulted in significantly increased hemolysis only when D5W as used (free hemoglobin, increased potassium); 2) EI trended lower when co-incubated with D5W and trended toward higher when co-incubated with prop; 3) aggregation was significantly lower when PRBCs co-incubated with NS, D5W, or Plasmalyte, and trended lower when co-incubated with epi, norepi, or dopa. Medication concentrations were between those predicted by distribution only in plasma and distribution through the entire intra- and extracellular space. CONCLUSION: Our data suggest that 5 minutes of PRBC incubation with isotonic crystalloids or catecholamines does not deleteriously alter PRBC hemolysis, membrane deformability, or aggregation. Co-incubation with D5W likely increases hemolysis. Propofol may promote hemolysis.

CD36 mediates H2O2-induced calcium influx in lung microvascular endothelial cells.

Elevated levels of reactive oxygen species and intracellular Ca2+ play a key role in endothelial barrier dysfunction in acute lung injury. We previously showed that H2O2-induced increases in intracellular calcium concentrations ([Ca2+]i) in lung microvascular endothelial cells (LMVECs) involve the membrane Ca2+ channel, transient receptor potential vanilloid-4 (TRPV4) and that inhibiting this channel attenuated H2O2-induced barrier disruption in vitro. We also showed that phosphorylation of TRPV4 by the Src family kinase, Fyn, contributes to H2O2-induced Ca2+ influx in LMVEC. In endothelial cells, Fyn is tethered to the cell membrane by CD36, a fatty acid transporter. In this study, we assessed the effect of genetic loss or pharmacological inhibition of CD36 on Ca2+ responses to H2O2 H2O2-induced Ca2+ influx was attenuated in LMVEC isolated from mice lacking CD36 (CD36-/-). TRPV4 expression and function was unchanged in LMVEC isolated from wild-type (WT) and CD36-/- mice, as well as mice with deficiency for Fyn (Fyn-/-). TRPV4 immunoprecipitated with Fyn, but this interaction was decreased in CD36-/- LMVEC. The amount of phosphorylated TRPV4 was decreased in LMVEC from CD36-/- mice compared with WT controls. Loss of CD36 altered subcellular localization of Fyn, while inhibition of CD36 fatty acid transport with succinimidyl oleate did not attenuate H2O2-induced Ca2+ influx. Lastly, we found that CD36-/- mice were protected from ischemia-reperfusion injury in vivo. In conclusion, our data suggest that CD36 plays an important role in H2O2-mediated lung injury and that the mechanism may involve CD36-dependent scaffolding of Fyn to the cell membrane to facilitate TRPV4 phosphorylation.

CD8(+)IL-17(+) T Cells Mediate Neutrophilic Airway Obliteration in T-bet-Deficient Mouse Lung Allograft Recipients.

Acute cellular rejection is a known risk factor for the development of obliterative bronchiolitis, which limits the long-term survival of lung transplant recipients. However, the T cell effector mechanisms in both of these processes remain incompletely understood. Using the mouse orthotopic lung transplant model, we investigated whether C57BL/6 T-bet(-/-) recipients of major histocompatibility complex (MHC)-mismatched BALB/c lung grafts develop rejection pathology and allospecific cytokine responses that differ from wild-type mice. T-bet(-/-) recipients demonstrated vigorous allograft rejection at 10 days, characterized by neutrophilic inflammation and predominantly CD8(+) T cells producing allospecific IL-17 and/or IFN-γ, in contrast to IFN-γ-dominant responses in WT mice. CD4(+) T cells produced IL-17 but not IFN-γ responses in T-bet(-/-) recipients, in contrast to WT controls. Costimulation blockade using anti-CD154 Ab significantly reduced allospecific CD8(+)IFN-γ(+) responses in both T-bet(-/-) and WT mice but had no attenuating effect on lung rejection pathology in T-bet(-/-) recipients or on the development of obliterative airway inflammation that occurred only in T-bet(-/-) recipients. However, neutralization of IL-17A significantly attenuated costimulation blockade-resistant rejection pathology and airway inflammation in T-bet(-/-) recipients. In addition, CXCL1 (neutrophil chemokine) was increased in T-bet(-/-) allografts, and IL-17 induced CXCL1 from mouse lung epithelial cells in vitro. Taken together, our data show that T-bet-deficient recipients of complete MHC-mismatched lung allografts develop costimulation blockade-resistant rejection characterized by neutrophilia and obliterative airway inflammation that is predominantly mediated by CD8(+)IL-17(+) T cells. Our data support T-bet-deficient mouse recipients of lung allografts as a viable animal model to study the immunopathogenesis of small airway injury in lung transplantation.

The Impact of Silent Liver Disease on Hospital Length of Stay Following Isolated Coronary Artery Bypass Grafting Surgery.

Objectives: Risk assessment models for cardiac surgery do not distinguish between degrees of liver dysfunction. We have previously shown that preoperative liver stiffness is associated with hospital length of stay following cardiac surgery. The authors hypothesized that a liver stiffness measurement (LSM) ≥ 9.5 kPa would rule out a short hospital length of stay (LOS < 6 days) following isolated coronary artery bypass grafting (CABG) surgery. Methods: A prospective observational study of one hundred sixty-four adult patients undergoing non-emergent isolated CABG surgery at a single university hospital center. Preoperative liver stiffness measured by ultrasound elastography was obtained for each participant. Multivariate logistic regression models were used to assess the adjusted relationship between LSM and a short hospital stay. Results: We performed multivariate logistic regression models using short hospital LOS (<6 days) as the dependent variable. Independent variables included LSM (< 9.5 kPa, ≥ 9.5 kPa), age, sex, STS predicted morbidity and mortality, and baseline hemoglobin. After adjusting for included variables, LSM ≥ 9.5 kPa was associated with lower odds of early discharge as compared to LSM < 9.5 kPa (OR: 0.22, 95% CI: 0.06-0.84, p = 0.03). The ROC curve and resulting AUC of 0.76 (95% CI: 0.68-0.83) suggest the final multivariate model provides good discriminatory performance when predicting early discharge. Conclusions: A preoperative LSM ≥ 9.5 kPa ruled out a short length of stay in nearly 80% of patients when compared to patients with a LSM < 9.5 kPa. Preoperative liver stiffness may be a useful metric to incorporate into preoperative risk stratification.

A case series evaluating the effect of esmolol therapy to treat hypoxemia in COVID-19 patients on VV-ECMO.

When COVID-19 ARDS abolishes pulmonary function, VV-ECMO can provide gas exchange. If oxygenation remains insufficient despite maximal VV-ECMO support, the addition of esmolol has been proposed. Conflict exists, however, as to the oxygenation level which should trigger beta-blocker initiation. We evaluated the effect of esmolol therapy on oxygenation and oxygen delivery in patients with negligible native lung function and various degrees of hypoxemia despite maximal VV-ECMO support. We found that, in COVID-19 patients with negligible pulmonary gas exchange, the generalized use of esmolol administration to raise arterial oxygenation by slowing heart rate and thereby match native cardiac output to maximal attainable VV ECMO flows actually reduces systemic oxygen delivery in many cases.

Reversing Neuromuscular Blockade without Nerve Stimulator Guidance in a Postsurgical ICU-An Observational Study.

We aimed to determine if not using residual neuromuscular blockade (RNB) analysis to guide neuromuscular blockade reversal administration in the postsurgical ICU resulted in consequences related to residual weakness. This single-center, prospective study evaluated 104 patients arriving in a postcardiac surgical ICU. After demonstrating spontaneous movement and T > 35.5 °C, all patients underwent RNB evaluation, and neostigmine/glycopyrrolate was then administered. When patients later demonstrated an adequate Rapid Shallow Breathing Index, negative inspiratory force generation, and arterial blood gas values with minimal mechanical ventilatory support, RNB evaluation was repeated in 94 of the 104 patients, and all patients were extubated. Though RNB evaluation was performed, patients were extubated without considering these results. Eleven of one hundred four patients had not achieved a Train-of-Four (TOF) count of four prior to receiving neostigmine. Twenty of ninety-four patients demonstrated a TOF ratio ≤ 90% prior to extubation. Three patients received unplanned postextubation adjunct respiratory support-one for obvious respiratory weakness, one for pain-related splinting compounding baseline disordered breathing but without obvious benefit from BiPAP, and one for a new issue requiring surgery. Residual neuromuscular weakness may have been unrecognized before extubation in 1 of 104 patients administered neostigmine without RNB analysis. ICU-level care may mitigate consequences in such cases.

Alveolar macrophages and Toll-like receptor 4 mediate ventilated lung ischemia reperfusion injury in mice.

BACKGROUND: Ischemia-reperfusion (I-R) injury is a sterile inflammatory process that is commonly associated with diverse clinical situations such as hemorrhage followed by resuscitation, transient embolic events, and organ transplantation. I-R injury can induce lung dysfunction whether the I-R occurs in the lung or in a remote organ. Recently, evidence has emerged that receptors and pathways of the innate immune system are involved in recognizing sterile inflammation and overlap considerably with those involved in the recognition of and response to pathogens. METHODS: The authors used a mouse surgical model of transient unilateral left pulmonary artery occlusion without bronchial involvement to create ventilated lung I-R injury. In addition, they mimicked nutritional I-R injury in vitro by transiently depriving cells of all nutrients. RESULTS: Compared with sham-operated mice, mice subjected to ventilated lung I-R injury had up-regulated lung expression of inflammatory mediator messenger RNA for interleukin-1ß, interleukin-6, and chemokine (C-X-C motif) ligand-1 and -2, paralleled by histologic evidence of lung neutrophil recruitment and increased plasma concentrations of interleukin-1ß, interleukin-6, and high-mobility group protein B1 proteins. This inflammatory response to I-R required toll-like receptor-4 (TLR4). In addition, the authors demonstrated in vitro cooperativity and cross-talk between human macrophages and endothelial cells, resulting in augmented inflammatory responses to I-R. Remarkably, the authors found that selective depletion of alveolar macrophages rendered mice resistant to ventilated lung I-R injury. CONCLUSIONS: The data reveal that alveolar macrophages and the pattern recognition receptor toll-like receptor-4 are involved in the generation of the early inflammatory response to lung I-R injury.

A Mouse Model of Orotracheal Intubation and Ventilated Lung Ischemia Reperfusion Surgery.

Ischemia reperfusion (IR) injury frequently results from processes that involve a transient period of interrupted blood flow. In the lung, isolated IR permits the experimental study of this specific process with continued alveolar ventilation, thereby avoiding the compounding injurious processes of hypoxia and atelectasis. In the clinical context, lung ischemia reperfusion injury (also known as lung IRI or LIRI) is caused by numerous processes, including but not limited to pulmonary embolism, resuscitated hemorrhagic trauma, and lung transplantation. There are currently limited effective treatment options for LIRI. Here, we present a reversible surgical model of lung IR involving first orotracheal intubation followed by unilateral left lung ischemia and reperfusion with preserved alveolar ventilation or gas exchange. Mice undergo a left thoracotomy, through which the left pulmonary artery is exposed, visualized, isolated, and compressed using a reversible slipknot. The surgical incision is then closed during the ischemic period, and the animal is awakened and extubated. With the mouse spontaneously breathing, reperfusion is established by releasing the slipknot around the pulmonary artery. This clinically relevant survival model permits the evaluation of lung IR injury, the resolution phase, downstream effects on lung function, as well as two-hit models involving experimental pneumonia. While technically challenging, this model can be mastered over the course of a few weeks to months with an eventual survival or success rate of 80%-90%.

Diazoxide preserves myocardial function in a swine model of hypothermic cardioplegic arrest and prolonged global ischemia.

OBJECTIVE: Adenosine triphosphate potassium sensitive channels provide endogenous myocardial protection via coupling of cell membrane potential to myocardial metabolism. Adenosine triphosphate potassium sensitive channel openers, such as diazoxide, mimic ischemic preconditioning, prevent cardiomyocyte swelling, preserve myocyte contractility after stress, and provide diastolic protection. We hypothesize that diazoxide combined with hyperkalemic cardioplegia provides superior myocardial protection compared with cardioplegia alone during prolonged global ischemia in a large animal model. METHODS: Twelve pigs were randomized to global ischemia for 2 hours with a single dose of cold blood (4:1) hyperkalemic cardioplegia alone (n = 6) or with diazoxide (500 µmol/L) (n = 6) and reperfused for 1 hour. Cardiac output, myocardial oxygen consumption, left ventricular developed pressure, left ventricular ejection fraction, diastolic function, myocardial troponin, myoglobin, markers of apoptosis, and left ventricular infarct size were compared. RESULTS: Four pigs in the cardioplegia alone group could not be weaned from cardiopulmonary bypass. There were no differences in myoglobin, troponin, or apoptosis between groups. Diazoxide preserved cardiac output versus control (74.5 vs 18.4 mL/kg/min, P = .01). Linear mixed regression modeling demonstrated that the addition of diazoxide to cardioplegia preserved left ventricular developed pressure by 36% (95% confidence interval, 9.9-61.5; P < .01), dP/dt max by 41% (95% confidence interval, 14.5-67.5; P < .01), and dP/dt min by 33% (95% confidence interval, 8.9-57.5; P = .01). It was also associated with higher (but not significant) myocardial oxygen consumption (3.7 vs 1.4 mL O2/min, P = .12). CONCLUSIONS: Diazoxide preserves systolic and diastolic ventricular function in a large animal model of prolonged global myocardial ischemia. Diazoxide as an adjunct to hyperkalemic cardioplegia may allow safer prolonged ischemic times during increasingly complicated cardiac procedures.

Selected Transesophageal Echocardiographic Parameters of Left Ventricular Diastolic Function Predict Length of Stay Following Coronary Artery Bypass Graft-A Prospective Observational Study.

(1) Importance: Abnormal left ventricular (LV) diastolic function, with or without a diagnosis of heart failure, is a common finding that can be easily diagnosed by intra-operative transesophageal echocardiography (TEE). The association of diastolic function with duration of hospital stay after coronary artery bypass (CAB) is unknown. (2) Objective: To determine if selected TEE parameters of diastolic dysfunction are associated with length of hospital stay after coronary artery bypass surgery (CAB). (3) Design: Prospective observational study. (4) Setting: A single tertiary academic medical center. (5) Participants: Patients with normal systolic function undergoing isolated CAB from September 2017 through June 2018. (6) Exposures: LV function during diastole, as assessed by intra-operative TEE prior to coronary revascularization. (7) Main Outcomes and Measures: The primary outcome was duration of postoperative hospital stay. Secondary intermediate outcomes included common postoperative cardiac, respiratory, and renal complications. (8) Results: The study included 176 participants (mean age 65.2 ± 9.2 years, 73% male); 105 (60.2%) had LV diastolic dysfunction based on selected TEE parameters. Median time to hospital discharge was significantly longer for subjects with selected parameters of diastolic dysfunction (9.1/IQR 6.6−13.5 days) than those with normal LV diastolic function (6.5/IAR 5.3−9.7 days) (p < 0.001). The probability of hospital discharge was 34% lower (HR 0.66/95% CI 0.47−0.93) for subjects with diastolic dysfunction based on selected TEE parameters, independent of potential confounders, including a baseline diagnosis of heart failure. There was a dose−response relation between severity of diastolic dysfunction and probability of discharge. LV diastolic dysfunction based on those selected TEE parameters was also associated with postoperative cardio-respiratory complications; however, these complications did not fully account for the relation between LV diastolic dysfunction and prolonged length of hospital stay. (9) Conclusions and Relevance: In patients with normal systolic function undergoing CAB, diastolic dysfunction based on selected TEE parameters is associated with prolonged duration of postoperative hospital stay. This association cannot be explained by baseline comorbidities or common post-operative complications. The diagnosis of diastolic dysfunction can be made by TEE.

Interleukin-6 mediates pulmonary vascular permeability in a two-hit model of ventilator-associated lung injury.

To test the hypothesis that interleukin-6 (IL-6) contributes to the development of ventilator-associated lung injury (VALI), IL-6-deficient (IL6(-/-)) and wild-type control (WT) mice received intratracheal hydrochloric acid followed by randomization to mechanical ventilation (MV + IT HCl) or spontaneous ventilation (IT HCl). After 4 hours, injury was assessed by estimation of lung lavage protein concentration and total and differential cell counts, wet/dry lung weight ratio, pulmonary cell death, histologic inflammation score (LIS), and parenchymal myeloperoxidase (MPO) concentration. Vascular endothelial growth factor (VEGF) concentration was measured in lung lavage and homogenate, as IL-6 and stretch both regulate expression of this potent mediator of permeability. MV-induced increases in alveolar barrier dysfunction and lavage VEGF were attenuated in IL6(-/-) mice as compared with WT controls, whereas tissue VEGF concentration increased. The effects of IL-6 deletion on alveolar permeability and VEGF concentration were inflammation independent, as parenchymal MPO concentration, LIS, and lavage total and differential cell counts did not differ between WT and IL6(-/-) mice following MV + IT HCl. These data support a role for IL-6 in promoting VALI in this two-hit model. Strategies to interfere with IL-6 expression or signaling may represent important therapeutic targets to limit the injurious effects of MV in inflamed lungs.

Incomplete Cushing's reflex in extracorporeal membrane oxygenation.

We report a case of intracranial hypertension presenting with bradycardia as the only component of Cushing's triad in a patient on extracorporeal membrane oxygenation. A 41-year-old woman with recurrent driveline infections of HeartMate-II had sternotomy and debridement that was complicated by right ventricular failure requiring veno-arterial extracorporeal membrane oxygenation. Patient was comatose and acute onset of bradycardia occurred without any change in blood pressure or respiration. Computed tomography of brain demonstrated an uncal herniation from diffuse cerebral edema. Acute onset of bradycardia in comatose patients may be the sole component of Cushing's triad in laminar flow circulatory support.

Chimerism, Transplant Tolerance, and Beyond.

The present review discusses current developments in tolerance induction for solid organ transplantation with a particular emphasis on chimerism-based approaches. It explains the basic mechanisms of chimerism-based tolerance and provides an update on ongoing clinical tolerance trials. The concept of "delayed tolerance" is presented, and ongoing preclinical studies in the nonhuman primate setting-including current limitations and hurdles regarding this approach-are illustrated. In addition, a brief overview and update on cell-based tolerogenic clinical trials is provided. In a critical approach, advantages, limitations, and potential implications for the future of these different regimens are discussed.

Strain-specific differences in sensitivity to ischemia-reperfusion lung injury in mice.

Ischemia-reperfusion (I/R) lung injury is characterized by increased pulmonary endothelial permeability and edema, but the genetic basis for this injury is unknown. We utilized an in vivo mouse preparation of unilateral lung I/R to evaluate the genetic determinants of I/R lung injury. An index of pulmonary vascular protein permeability was measured by the ratio of left-to-right lung Evans blue dye of eight inbred mouse strains after 30 min of left lung ischemia and 150 min of reperfusion. The order of strain-specific sensitivity to I/R lung injury was BALB/c < SJL/J < CBA/J < C57BL/6J < 129/J < A/J < C3H/H3J < SWR/J. The reciprocal F1 offspring of the BALB/c and SWR/J progenitor strains had intermediate phenotypes but a differing variance. A similar pattern of right lung Evans blue dye content suggested the presence of contralateral injury because baseline vascular permeability was not different. Lung I/R injury was attenuated by NADPH oxidase inhibition, indicating a role for NADPH oxidase-derived reactive oxygen species (ROS). There was no strain-dependent difference in lung NADPH oxidase expression. Strain-related differences in zymosan-stimulated neutrophil ROS production did not correlate with I/R lung injury in that neutrophil ROS production in SWR/J mice was greater than C57BL/6J but not different from BALB/c mice. These data indicate the presence of a genetic sensitivity to lung I/R injury that involves multiple genes including a maternal-related factor. Although neutrophil-derived ROS production is also modulated by genetic factors, the pattern did not explain the genetic sensitivity to lung I/R injury.

Induction of Major Histocompatibility Complex-mismatched Mouse Lung Allograft Acceptance With Combined Donor Bone Marrow: Lung Transplant Using a 12-Hour Nonmyeloablative Conditioning Regimen.

BACKGROUND: Despite broad and intense conventional immunosuppression, long-term survival after lung transplantation lags behind that for other solid organ transplants, primarily because of allograft rejection. Therefore, new strategies to promote lung allograft acceptance are urgently needed. The purpose of the present study was to induce allograft tolerance with a protocol compatible with deceased donor organ utilization. METHODS: Using the major histocompatibility complex-mismatched mouse orthotopic lung transplant model, we investigated a conditioning regimen consisting of pretransplant T cell depletion, low-dose total body irradiation and posttransplant (donor) bone marrow, and splenocyte infusion followed by posttransplantation cyclophosphamide. RESULTS: Our results show that C57BL/6 recipients of BALB/c lung allografts undergoing this complete short-duration nonmyeloablative conditioning regimen had durable lung allograft acceptance. Mice that lacked 1 or more components of this regimen exhibited significant graft loss. Mechanistically, animals with lung allograft acceptance had established higher levels of donor chimerism, lymphocyte responses which were attenuated to donor antigens but maintained to third-party antigens, and clonal deletion of donor-reactive host Vß T cells. Frequencies of Foxp3 T regulatory cells were comparable in both surviving and rejected allografts implying that their perturbation was not a dominant cell-regulatory mechanism. Donor chimerism was indispensable for sustained tolerance, as evidenced by acute rejection of allografts in established chimeric recipients of posttransplantation cyclophosphamide after a chimerism-ablating secondary recipient lymphocyte infusion. CONCLUSIONS: Together, these data provide proof-of-concept for establishing lung allograft tolerance with tandem donor bone marrow transplantation using a short-duration nonmyeloablative conditioning regimen and posttransplant cyclophosphamide.

Effect of ischemia and reperfusion without airway occlusion on vascular barrier function in the in vivo mouse lung.

Ischemia-reperfusion (I/R) lung injury causes increased vascular permeability and edema. We developed an in vivo murine model of I/R allowing measurement of pulmonary vascular barrier function without airway occlusion. The left pulmonary artery (PA) was occluded with an exteriorized, slipknotted suture in anesthetized C57BL/6J mice. The effect of ischemic time was determined by subjecting mice to 5, 10, or 30 min of left lung ischemia followed by 150 min of reperfusion. The effect of reperfusion time was determined by subjecting mice to 30 min of left lung ischemia followed by 30 or 150 min of reperfusion. Changes in pulmonary vascular barrier function were measured with the Evans blue dye (EBD) technique, dual-isotope radiolabeled albumin (RA), bronchoalveolar lavage (BAL) protein concentration, and wet weight-to-dry weight ratio (WW/DW). Increasing left lung ischemia with constant reperfusion time or increasing left lung reperfusion time after constant ischemic time resulted in significant increases in left lung EBD content at all times compared with both right lung values and sham surgery mice. The effects of left lung ischemia on lung EBD were corroborated by RA but the effects of increasing reperfusion time differed, suggesting binding of EBD to lung tissue. An increase in WW/DW was only detected after 30 min of reperfusion, suggesting edema clearance. BAL protein concentrations were unaffected. We conclude that short periods of I/R, without airway occlusion, increase pulmonary vascular permeability in the in vivo mouse, providing a useful model to study molecular mechanisms of I/R lung injury.

Interactive effects of mechanical ventilation and kidney health on lung function in an in vivo mouse model.

We hypothesized that the influence of acute kidney injury (AKI) on the sensitivity of the lung to an injurious process varies with the severity of the injurious process. Thus, we thought that AKI would exacerbate lung injury from low degrees of lung trauma but attenuate lung injury from higher degrees of lung trauma. C57BL/6 mice underwent AKI (30-min kidney ischemia) or sham surgery, followed at 24 h by 4 h of spontaneous breathing (SB), mechanical ventilation with low tidal volume (7 ml/kg, LTV), or mechanical ventilation with high tidal volume (30 ml/kg, HTV). Compared with LTV, median bronchoalveolar lavage (BAL) protein leak was significantly lower with SB and greater with HTV in both sham and AKI mice. Compared with LTV, median Evans blue dye-labeled albumin extravasation in lungs (L-EBD) was also significantly lower with SB and greater with HTV. L-EBD showed a significant interaction between ventilatory mode and kidney health, such that AKI attenuated the L-EBD rise seen in HTV vs. LTV sham mice. An interaction between ventilatory mode and kidney health could also be seen in BAL neutrophil number (PMN). Thus, AKI attenuated the BAL PMN rise seen in HTV vs. LTV sham mice. These data support the presence of a complex interaction between mechanical ventilation and AKI in which the sensitivity of the lung to trauma varies with the magnitude of the trauma and may involve a modification of pulmonary neutrophil activity by AKI.