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BACKGROUND: Esophageal adenocarcinoma (EA) is a typical immunogenic malignant tumor with a dismal 5-year survival rate lower than 20%. Although miRNA-3648 (miR-3648) is expressed abnormally in EA, its impact on the tumor immune microenvironment remains unknown. In this study, we sought to identify immune-related genes (IRGs) that are targeted by miR-3648 and develop an EA multigene signature. METHODS: The gene expression data of 87 EA tumor samples and 67 normal tissue samples from The Cancer Genome Atlas (TCGA) database and the Genotype-Tissue Expression (GTEx) database were downloaded, respectively. Weighted gene co-expression network analysis (WGCNA), the CIBERSORT algorithm, and Cox regression analysis were applied to identify IRGs and to construct a prognostic signature and nomogram. RESULTS: MiR-3648 was expectedly highly expressed in EA tumor tissues (P=2.6e-8), and related to the infiltration of activated natural killer cells (NK cells) and activated CD4 T lymphocytes (CD4 cells). A total of 70 miR-3648-targeted genes related to immune cell infiltration were identified. Among them, 4 genes (C10orf55, DLL4, PANX2, and NKAIN1) were closely related to overall survival (OS), and were thus selected to construct a 4-gene risk score (RS). The RS had a superior capability to predict OS [area under the curve (AUC) =0.740 for 1 year; AUC =0.717 for 3 years; AUC =0.622 for 5 years]. A higher score was indicative of a poorer prognosis than a lower score [hazard ratio (HR) =2.71; 95% confidence interval (CI): 1.45–5.09; P=0.002]. Furthermore, the nomogram formed by combining the RS and the TNM classification of malignant tumors (TNM stage) improved the accuracy of survival prediction [Harrell’s concordance index (C-index) =0.698]. CONCLUSIONS: MiR-3648 may play a critical role in EA pathogenesis. The novel 4-gene signature may serve as a prognostic tool to manage patients with EA.

Introduction Some patients with cardiac stents will need thoracic surgery during the dual antiplatelet therapy (DAPT) period. When surgery cannot be safely delayed to allow 1 year of uninterrupted DAPT, appropriate perioperative management of anticoagulation is critical. Case presentation A patient treated with new drug-eluting stents and DAPT was concomitantly diagnosed with lung cancer and required a lobectomy. We describe the novel addition of ticagrelor (a short-acting oral antiplatelet agent) to eptifibatide (a short-acting intravenous antiplatelet agent) to bridge DAPT for surgery. Discussion This ticagrelor-eptifibatide perioperative bridge resulted in decreased preoperative hospitalization compared with eptifibatide alone. There were no associated perioperative cardiac or bleeding complications.

Mucor is a ubiquitous fungus that is non-pathogenic in healthy people. In immunocompromised hosts, nonfunctional or absent neutrophils and macrophages result in fungal invasion and infection [1]. Invasive mucor (mucormycosis) most commonly involves the sinuses, brain, or lungs. Pulmonary mucormycosis typically presents in patients with a history of organ transplantation or hematologic malignancy [2], and is rare in patients with diabetes alone. The epidemiology and management of pediatric pulmonary mucormycosis is poorly described. We report an unusual occurrence of this disease, complicated by segmental pulmonary artery thrombus in a 15-year-old with poorly controlled diabetes. His severe, medication-resistant infection was ultimately treated successfully with antifungal medication combined with aggressive surgical debridement. The pulmonary artery segmental thrombus resolved after treatment of the underlying infection without anticoagulation. Keywords: Mucormycosis, Pulmonary mucormycosis, Diabetes, Pneumonectomy, Clagett procedure, Pediatric thoracic surgery

Context Progel Pleural Air Leak Sealant (CR Bard, Warwick, RI) is a US Food and Drug Administration-approved hydrogel designed for application to surgical staple lines to prevent air leak after lung surgery. This product has demonstrated efficacy in reducing intraoperative air leaks compared with standard air leak closure methods. However, the impact on chest tube duration and length of hospital stay has not been reported. Objective To evaluate the effect on rates of postoperative air leak, chest tube duration, and hospital stay in surgical patients with and without use of Progel. Design Retrospective study of 176 patients aged 18 to 80 years who underwent video-assisted thoracoscopic wedge resections between 2014 and 2016. Eighty-four (48%) cases using Progel were included, as well as a representative sample of non-Progel cases (n = 92; 52%). Main outcome measure Presence of postoperative lung air leak. Results No difference existed between the Progel and non-Progel groups in the rate of postoperative air leak (20/84, 23.81% Progel; 16/92, 17.39% non-Progel; p = 0.33). The length of time patients had a chest tube was similar (23.5 vs 23 hours, p = 0.721), as was percentage of patients with a less than 2-day hospitalization (77.17% non-Progel vs 82.14% Progel, p = 0.414). Conclusion Our results suggest that Progel, used routinely in patients undergoing nonanatomic lung resection, does not have a significant impact on postoperative air leak, chest tube duration, or length of hospital stay. Further studies are warranted to evaluate the utility of Progel in reducing postoperative complications after thoracoscopic wedge resection in those treated for air leak or in the reduction of postoperative air leak in high-risk patients.

Current literature favors a volume-outcome relationship in pulmonary lobectomy that prompted centralization of these operations abroad, in national, single-payer health care settings. This study examined the impact of regionalization on outcomes after lung cancer resection within a US integrated health care system.This study retrospectively reviewed major pulmonary resections (lobectomy, bilobectomy, pneumonectomy) for lung cancer that were performed before (2011 to 2013; n = 782) and after (2015 to 2017; n = 845) thoracic surgery regionalization during 2014.Case migration from 16 regionwide sites to 5 designated centers was complete by 2016. Facility volume increased from 17.4 to 48.3 cases/y (P = .002), and surgeon volume increased from 12.5 to 19.9 cases/y (P = .001). The postregionalization era was characterized by increased video-assisted thoracoscopic surgery (86% from 57%; P.001), as well as decreased intensive care unit use (-1.0 days; P.001) and hospital length of stay (-3.0 days; P.001). Postregionalization patients experienced fewer total (26.2% from 38.6%; P.001) and major (9.6% from 13.6%; P = .01) complications. The association between regionalization and decreased length of stay and morbidity was independent of surgical approach and case volume in mixed multivariate models.After the successful implementation of thoracic surgery regionalization in our US health care network, pulmonary resection volume increased, and practice shifted to majority video-assisted thoracoscopic surgery and minimum intensive care unit utilization. Regionalization was independently associated with significant reductions in length of stay and morbidity.

Tietze syndrome is a rare and benign cause of chest pain that may be confused with life-threatening conditions. Characteristic findings are painful, localized inflammation of the costosternal, sternoclavicular, or costochondral joints typically of the second and third ribs. We present a patient with severe, medically refractory symptoms consistent with Tietze syndrome. Although surgical management is not the typical treatment course, symptomatic control was eventually achieved with complete resection of the involved cartilage and adjacent rib. We propose surgical management in patients with debilitating cases of Tietze syndrome that are resistant to conservative therapies.

Postsurgical pain control can have a significant impact on patient outcomes and hospital-associated costs. We sought to evaluate the effect of intercostal nerve blocks using liposomal bupivacaine on length of stay (LOS) in patients undergoing video-assisted thoracoscopic surgery (VATS).We retrospectively reviewed outcomes in 80 patients undergoing VATS wedge resection, VATS lobectomy, or minimally-invasive esophagectomy (MIE). Patients received either liposomal bupivacaine (n=40) or standard-release bupivacaine with epinephrine (n=40) via intercostal nerve block. The LOS, 24-hour postoperative pain scores, overall opioid usage, and patient ambulation rates at 24 hours were compared for the two groups.The median LOS was significantly shorter in patients receiving liposomal bupivacaine, at 1.35 days (IQR, 1.28 to 1.53 days) compared to 2.45 days (IQR, 2.08 to 3.51 days) in patients receiving standard-release bupivacaine (P0.0001). Average post-operative pain score during the first 24 hours was 3.4±1.8 for the liposomal bupivacaine group and 2.3±1.2 for the control group (P=0.002). This difference, though statistically significant, is likely not clinically significant, as there was no difference in 24-hour postoperative intravenous morphine equivalent usage between the liposomal bupivacaine and control groups (29.8±21.0The use of liposomal bupivacaine is associated with decreased LOS in postoperative thoracic surgery patients and earlier return to ambulation. It does not, however, decrease 24-hour postoperative pain scores or opioid usage.

Recurrent respiratory papillomatosis (RRP) is a disease of the respiratory tract caused by infection with the human papillomavirus (HPV) and is characterized by multiple recurring papillomas throughout the respiratory tract. Although rare, extra laryngeal involvement carries the risk of malignant transformation in 3–7% of adults. We report the case of a patient with unmonitored juvenile onset RRP with pulmonary involvement found to have malignant transformation to squamous cell carcinoma (SCC). Incidentally found on chest radiography for mild chest wall trauma, she was found to have a large left lower lobe mass with pathology consistent with SCC. The patient underwent surgical management with thoracoscopic left lower lobectomy and mediastinal lymph node dissection followed by adjuvant chemoradiation for pathologic stage IIIA SCC. Surveillance imaging at 6 months shows no evidence of disease progression.

INTRODUCTION Tracheoinnominate fistula, a rare complication of tracheostomy, carries high mortality regardless of treatment; therefore prevention and quick diagnosis is pertinent to survival. CASE PRESENTATION A 76-year-old man who underwent emergent tracheostomy placement presented on postoperative day 10 with massive hemorrhage concerning for tracheoinnominate fistula and was treated with median sternotomy and ligation of the innominate artery. DISCUSSION This presentation describes a concise diagnosis and treatment plan for a rare event. The key to good outcomes is quick diagnosis and urgent surgical intervention.

Background IL-16 promotes the recruitment of various cells expressing CD4, a receptor for IL-16. The precise role of IL-16 in transplant rejection remains unknown; therefore, the present study investigated the contribution of IL-16 to the development of chronic rejection in heart transplants. Methods C-H-2 bm12 KhEg (H-2 bm12 ) donor hearts were transplanted into (1) IL-16-deficient (IL-16 −/− ) C57BL/6J or (b) wild type (WT) control recipients (MHC class II mismatch). Grafts were harvested at 52 days, parenchymal rejection was assessed by the ISHLT grading system, and CAV was examined morphometrically. Graft infiltrating cells were detected 10 and 52 days after transplantation. Intragraft cytokine and chemokine profiles were assessed. To confirm the role of IL-16 in CAV development, C-H-2 bm12 KhEg (H-2 bm12 ) donor hearts were transplanted into C57BL/6J WT recipients treated with (1) anti-IL-16-neutralization monoclonal antibody or (b) control immunoglobulin G. Grafts were harvested at 52 days, and CAV was quantified morphometrically. Graft-infiltrating cells were examined histologically. Results Parenchymal rejection and CAV was significantly attenuated in donor hearts transplanted into IL-16 −/− recipient mice compared with WT controls. Donor hearts transplanted into IL-16 −/− recipients had a significant reduction in coronary artery luminal occlusion, intima-to-media ratio, and percentage of diseased vessels. CAV was associated with decreased donor organ inflammation, as well as donor organ cytokine (IL-1β and IL-6) and chemokine (MCP-1 and KC) protein expression. Intimal proliferation and inflammatory cell infiltration were significantly reduced in hearts transplanted into recipients treated with an IL-16-neutralization antibody. Conclusions IL-16-deficiency reduced graft inflammatory cell recruitment, and allograft inflammatory cytokine and chemokine production. Therefore, IL-16 neutralization may provide a potential target for novel therapeutic treatment for cardiac allograft rejection.

This study investigates the protective effect of exogenous αB-crystallin (CryAB) on myocardial function after ischemia-reperfusion injury.Mice underwent temporary left anterior descending artery occlusion for 30 minutes. Either CryAB (50 μg) or phosphate-buffered saline (100 μL [n=6, each group]) were injected in the intramyocardial medial and lateral perinfarct zone 15 minutes before reperfusion. Intraperitoneal injections were administered every other day. Left ventricular ejection fraction was evaluated on postoperative day 40 with magnetic resonance imaging. To investigate the effect of CryAB on apoptosis after hypoxia/reoxygenation in vitro, murine atrial cardiomyocytes (HL-1 cells) or human microvascular endothelial cells (HMEC-1) were incubated with either 50 μg CryAB (500 μg /10 mL) or phosphate-buffered saline in a hypoxia chamber for 6, 12, and 24 hours, followed by 30 minutes of reoxygenation at room air. Apoptosis was then assessed by western blot (Bcl-2, free bax, cleaved caspases-3, 9, PARP) and enzyme-linked immunosorbent assay analyses (cytoplasmic histone-associated DNA fragments and caspase-3 activity).On postoperative day 40, CryAB-treated mice had a 1.8-fold increase in left ventricular ejection fraction versus control mice (27%±6% versus 15%±4% SD, p0.005). In vitro, (1) the HL-1 cells showed no significant difference in apoptotic protein expression, cytoplasmic histone-associated DNA fragments, or caspase-3 activity; (2) the HMEC-1 cells had increased but not significant apoptotic protein expression with, however, a significant decrease in cytoplasmic histone-associated DNA fragments (1.5-fold, p0.01) and caspase-3 activity (2.7-fold, p0.005).Exogenous CryAB administration significantly improves cardiac function after ischemia-reperfusion injury, in vivo. The protective anti-apoptotic affects of CryAB may target the endothelial cell.

Background Although interleukin-17 (IL-17) has been reported to participate in the pathogenesis of infectious, autoimmune and allergic disorders, the precise role in allograft rejection remains uncertain. This study illustrates that IL-17 contributes to the pathogenesis of chronic allograft rejection. Result Utilizing a murine heterotopic heart transplant model system, IL-17-deficient recipient mice had decreased allograft inflammatory cell recruitment, decreased IL-6, MCP-1, and KC production, and reduced graft coronary artery disease (GCAD). Intragraft gamma delta (γδ) T cells appear to be the predominant source of IL-17 production. Conclusion Therefore, IL-17 neutralization may provide a potential target for novel therapeutic treatment for cardiac allograft rejection.

Left atrial-esophageal fistula (LAEF) is a rare complication of radiofrequency ablation (RFA) procedures undertaken for atrial fibrillation (AF). This complication is associated with significant morbidity and mortality. Currently, there is no clear consensus on the appropriate management strategy. We report a case of a LAEF that developed in a patient 2 weeks after RFA for medication refractory AF. The patient underwent successful repair of the fistula through a left posterolateral thoracotomy, wherein the esophageal and atrial lesions were repaired primarily with an intercostal muscle flap and bovine pericardial patch to reinforce and prevent recurrence.

Three α-subunit isoforms of the sodium pump, which is the receptor for cardiac glycosides, are expressed in human heart. The aim of this study was to determine whether these isoforms have distinct affinities for the cardiac glycoside ouabain. Equilibrium ouabain binding to membranes from a panel of different human tissues and cell lines derived from human tissues was compared by an F statistic to determine whether a single population of binding sites or two populations of sites with different affinities would better fit the data. For all tissues, the single-site model fit the data as well as the two-site model. The mean equilibrium dissociation constant ( Kd) for all samples calculated using the single-site model was 18 ± 6 nM (mean ± SD). No difference in Kdwas found between nonfailing and failing human heart samples, although the maximum number of binding sites in failing heart was only ∼50% of the number of sites in nonfailing heart. Measurement of association rate constants and dissociation rate constants confirmed that the binding affinities of the different human α-isoforms are similar to each other, although calculated Kdvalues were lower than those determined by equilibrium binding. These results indicate both that the affinity of all human α-subunit isoforms for ouabain is similar and that the increased sensitivity of failing human heart to cardiac glycosides is probably due to a reduction in the number of pumps in the heart rather than to a selective inhibition of a subset of pumps with different affinities for the drugs.

Urea transporters have been cloned from kidney medulla (UT-A) and erythrocytes (UT-B). We determined whether UT-A proteins could be detected in heart and whether their abundance was altered by uremia or hypertension or in human heart failure. In normal rat heart, bands were detected at 56, 51, and 39 kDa. In uremic rats, the abundance of the 56-kDa protein increased 1.9-fold compared with pair-fed, sham-operated rats, whereas the 51- and 39-kDa proteins were unchanged. We also detected UT-A2 mRNA in hearts from control and uremic rats. Because uremia is accompanied by hypertension, the effects of hypertension per se were studied in uninephrectomized deoxycorticosterone acetate salt–treated rats, where the abundance of the 56-kDa protein increased 2-fold versus controls, and in angiotensin II–infused rats, where the abundance of the 56 kDa protein increased 1.8-fold versus controls. The 51- and 39-kDa proteins were unchanged in both hypertensive models. In human left ventricle myocardium, UT-A proteins were detected at 97, 56, and 51 kDa. In failing left ventricle (taken at transplant, New York Heart Association class IV), the abundance of the 56-kDa protein increased 1.4-fold, and the 51-kDa protein increased 4.3-fold versus nonfailing left ventricle (donor hearts). We conclude that (1) multiple UT-A proteins are detected in rat and human heart; (2) the 56-kDa protein is upregulated in rat heart in uremia or models of hypertension; and (3) the rat results can be extended to human heart, where 56- and 51-kDa proteins are increased during heart failure.

Background— Interleukin-17 (IL-17), which is predominantly produced by T helper 17 cells distinct from T helper 1 or T helper 2 cells, participates in the pathogenesis of infectious, autoimmune, and allergic disorders. However, the precise role in allograft rejection remains uncertain. In the present study, we investigated the role of IL-17 in acute allograft rejection using IL-17-deficient mice. Methods and Results— Donor hearts from FVB mice were heterotopically transplanted into either C57BL/6J-IL-17-deficient (IL-17 − / − ) or -wild-type mice. Allograft survival was significantly prolonged in IL-17 − / − recipient mice due to reduced local inflammation accompanied by decreased inflammatory cell recruitment and cytokine/chemokine expression. IL-17 − / − recipient mice exhibited decreased IL-6 production and reciprocally enhanced regulatory T cell expansion, suggesting a contribution of regulatory T cells to prolonged allograft survival. Indeed, allografts transplanted into anti-CD25 mAb-treated IL-17 − / − recipient mice (regulatory T cell-depleted) developed acute rejection similar to wild-type recipient mice. Surprisingly, we found that gamma delta T cells rather than CD4 + and CD8 + T cells were key IL-17 producers in the allografts. In support, equivalent allograft rejection was observed in Rag-2 −/− recipient mice engrafted with either wild-type or IL-17 − / − CD4 + and CD8 + T cells. Finally, hearts transplanted into gamma delta T cell-deficient mice resulted in decreased allograft rejection compared with wild-type controls. Conclusions— During heart transplantation, (1) IL-17 is crucial for acceleration of acute rejection; (2) IL-17-deficiency enhances regulatory T cell expansion; and (3) gamma delta T cells rather than CD4 + and CD8 + T cells are a potential source of IL-17. IL-17 neutralization may provide a potential target for novel therapeutic treatment for cardiac allograft rejection.

Background In this study we systematically dissect the prenylation pathway to better define the mechanism behind statin inhibition in chronic allograft rejection in heart transplants, or transplant coronary artery disease (TCAD). Methods Utilizing a murine heterotopic heart transplant model, animals received daily treatments of either statin or selective isoprenoid blockade inhibitors to block the four major downstream branches of the mevalonate pathway. TCAD was assessed by morphometric analysis at Day 52. Graft-infiltrating cells, cytokine production, smooth muscle cell proliferation and migration and endothelial cell MHC II expression were detected on Day 7. Results Atorvastatin and two prenylation inhibitors, NE-10790 and manumycin A, significantly reduced TCAD lesions compared with untreated animals. Perillyl alcohol treatment resulted in a trend toward decreased luminal narrowing. Finally, zaragozic acid (cholesterol blockade only) did not alter TCAD severity. Statins and prenylation inhibitors reduced inflammatory cell allograft recruitment, but did not always correlate with TCAD reduction. Cytokine production was decreased in recipient spleens in all treatment groups. Both in vitro and in vivo IFN-γ–stimulated MHC II expression was decreased in a dose-dependent manner in the atorvastatin, perillyl alcohol and NE-10790 groups. In vitro smooth muscle cell proliferation was decreased in all treatment groups. Finally, in vitro smooth muscle cell migration was decreased in the atorvastatin, NE-10790 and manumycin A groups only. Conclusions FPT and GGPT-2 (inhibition) are the key enzymes in the HGM-CoA reductase pathway and most influential in TCAD prevention. TCAD reduction is most closely related to smooth muscle cell migration, but not its anti-inflammatory properties.

Background Mast cells are hypothesized to promote rejection and adverse remodeling in cardiac allografts. In contrast, it has been reported that mast cells may enhance cardiac allograft survival in rats. We used C57BL/6- Kit W-sh/W-sh mast cell-deficient and corresponding wild-type mice to investigate possible contributions of recipient mast cells to acute or chronic cardiac allograft rejection. Methods FVB (H-2 q ; acute rejection), or C-H-2 bm12 KhEg (H-2 bm12 ; chronic rejection) donor hearts were heterotopically transplanted into C57BL/6- Kit W-sh/W-sh (H-2 b ) or C57BL/6- Kit +/+ (H-2 b ) mice. The degree of acute rejection was assessed at 5 days and chronic rejection, at 52 days. Results In the acute rejection model, donor heart vascular cell adhesion molecule-1 (VCAM-1) expression was significantly lower in C57BL/6- Kit W-sh/W-sh than in wild-type recipients; however, acute rejection scores, graft survival, inflammatory cells, or cytokine expression did not differ significantly. In the chronic rejection model, the number of mast cells/mm 2 of allograft tissue was significantly increased 52 days after transplantation in allografts transplanted into C57BL/6- Kit +/+ but not C57BL/6- Kit W-sh/W-sh mice; however, no substantial differences were noted in graft coronary artery disease, graft inflammatory cells, or levels of graft tissue expression of cytokines or adhesion molecules. Conclusions Cardiac allografts undergoing chronic rejection in wild-type C57BL/6- Kit +/+ mice exhibit increased numbers of mast cells, but acute or chronic cardiac allograft rejection can develop in C57BL/6- Kit W-sh/W-sh mice even though these recipients virtually lack mast cells. These findings indicate that recipient mast cells are not required for acute or chronic cardiac allograft rejection in the models examined.

BACKGROUND This study aimed at investigating the role of a novel JAK3 inhibitor, R348, in the prevention of chronic airway allograft rejection. METHODS The heterotopic rat trachea transplant model was used. Recipients were treated daily with R348 (10, 20, 40, 80 mg/kg) or rapamycin (0.75 or 3 mg/kg). Blood levels of R348 and of its active metabolite R333 were measured. Grafts were harvested after 28 days to analyze epithelial morphology, mononuclear infiltration, and luminal obliteration. Plasma levels of circulating donor strain-reactive IgG antibodies were quantified. RESULTS R348 was well tolerated at up to 40 mg/kg, but was toxic at 80 mg/kg. Blood levels of R333 at 2 and 24 hr were consistently 10 to 15 times higher than those of R348. Airway luminal obliteration after 28 days was significantly inhibited by R348 at 40 mg/kg (20.6%+/-13.2%, P

Mesenchymal stem cells hold promise for cardiovascular regenerative therapy. Derivation of these cells from the adipose tissue might be easier compared with bone marrow. However, the in vivo fate and function of adipose stromal cells (ASC) in the infarcted heart has never been compared directly to bone marrow-derived mesenchymal cells (MSC).ASC and MSC were isolated from transgenic FVB mice with a beta-actin promoter driving firefly luciferase and green fluorescent protein double fusion reporter gene, and they were characterized using flow cytometry, microscopy, bioluminescence imaging and luminometry. FVB mice (n=8 per group) underwent myocardial infarction followed by intramyocardial injection of 5x10(5) ASC, MSC, fibroblasts (Fibro, positive control), or saline (negative control). Cell survival was measured using bioluminescence imaging for 6 weeks and cardiac function was monitored by echocardiography and pressure-volume analysis. Ventricular morphology was assessed using histology.ASC and MSC were CD34(-), CD45(-), c-Kit(-), CD90(+), Sca-1(+), shared similar morphology and had a population doubling time of approximately 2 days. Cells expressed Fluc reporter genes in a number-dependent fashion as confirmed by luminometry. After cardiac transplantation, both cell types showed drastic donor cell death within 4 to 5 weeks. Furthermore, transplantation of either cell type was not capable of preserving ventricular function and dimensions, as confirmed by pressure-volume-loops and histology.This is the first study comparing the in vivo behavior of both cell types in the infarcted heart. ASC and MSC do not tolerate well in the cardiac environment, resulting in acute donor cell death and a subsequent loss of cardiac function similar to control groups.

Background Janus kinase (JAK)3 is crucial for signal transduction downstream of various cytokine receptors in immune cells. This is the first report on the novel JAK3 inhibitor R348. Methods (1) Detailed pharmacokinetic data were obtained in rats; (2) multiple in vitro enzyme inhibition assays were performed to characterize the drug; (3) prevention of acute rejection was investigated in animals treated with different doses of R348 or rapamycin for 5 days; and (4) cardiac allograft survival after a 10-day treatment period was studied for various regimens of R348, tacrolimus, or rapamycin; combination indices were calculated to evaluate drug interactions. Results (1) Plasma levels of R348's active metabolite R333 sustained high for 8 hr or more, depending on the dose. (2) In vitro enzyme assays showed potent inhibition of JAK3- and Syk-dependent pathways. (3) R348 40 mg/kg preserved graft function, significantly reduced graft infiltration, and decreased histologic ISHLT rejection scores on postoperative day 5. Results were similar to those of rapamycin 3 mg/kg. Likewise, both drugs significantly reduced the cellular Th1 and Th2 immune responses, as determined by enzyme-linked immunosorbent assays. Intragraft inflammatory cytokine upregulation was similarly suppressed by R348 and rapamycin. R348 10 mg/kg was subtherapeutic. (4) Allograft survival was similar for R348 20 and 40 mg/kg, which was comparable with therapeutically dosed tacrolimus or rapamycin. In combination regimens, R348 demonstrated highly beneficial synergistic interactions with tacrolimus. Conclusions R348 is a promising novel JAK3/Syk-inhibitor with favorable pharmacokinetics and biological activity. It effectively diminishes acute cardiac allograft rejection and is suitable for combination regimens with tacrolimus.

Cardiac sodium pumps (Na,K-ATPase) influence cell calcium and contractility by generating the Na+ gradient driving Ca++ extrusion via the Na+/Ca++ exchanger (NCX), and are the receptors for cardiac glycosides such as digitalis which increases cardiac contractility by decreasing the Na+ gradient driving Ca++ extrusion. There are multiple isoforms of the sodium pump expressed in the heart indicating the potential for isoform specific expression patterns, function and regulation. Regarding isoform expression patterns, human heart expresses alpha1, alpha2, alpha3, beta1 and a small amount of beta2. Within the human heart, alpha3, beta1 and NCX levels are 30-50% lower in atria than ventricles, associated with increased sensitivity to inotropic stimulation. Distribution at the cellular level has been studied in the rat heart where both alpha1 and alpha2 are detected in the T-tubules along with NCX. Regarding isoform function, we focussed on human sodium pumps as cardiac glycoside receptors. A study of human sodium pump expressed alone (alpha1) or in combination (alpha1 with alpha2, or alpha1 with alpha2 and alpha3) in their native membranes aimed to determine whether different isoforms had distinct affinities for the cardiac glycoside ouabain by evaluating whether the ouabain binding data were best fit with a single site or two site model. The results indicated that the affinity of these human a subunit isoforms for ouabain is indistinguishable, and that changes in sensitivity to cardiac glycosides during heart failure are likely due to a decrease in the total number of pumps rather than a shift in expression to a more sensitive isoform. Regarding isoform regulation, we hypothesized that a primary decrease in cardiac Na,K-ATPase expression would be associated with a secondary increase in cardiac Na+/Ca++ exchanger expression as a homeostatic mechanism to blunt an increase in cell Ca++ stores (and visa versa with an increase in Na,K-ATPase). Supporting the hypothesis: in a rat model of renovascular hypertension, or after treatment with amiodarone there are 50% decreases in alpha2 levels with 35-40% increases in NCX levels in left ventricle, while in the transition from hypo- to hyperthyroid, there are increases in both alpha1 (2-fold) and alpha2 (8-fold) with decreases in NCX (0.45-fold). In comparison, in transgenic mice overexpressing NCX, there was no secondary change in Na,K-ATPase alpha1 or alpha2 levels indicating that primary changes in NCX do not drive secondary changes in Na,K-ATPase in the heart. This information provides the basis for addressing the significant gaps in our understanding of the physiologic, structural and homeostatic coupling between sodium pump isoforms and Na+/Ca++ exchangers in the heart and how coupling is related to control of cardiac contractility in health and disease.