Association of antibiotics with the outcomes in COVID-19 pneumonia patients with elevated PCT levels.

OBJECTIVE: To assess antibiotics impact on outcomes in COVID-19 pneumonia patients with varying procalcitonin (PCT) levels. METHODS: This retrospective cohort study included 3665 COVID-19 pneumonia patients hospitalized at five Mayo Clinic sites (March 2020 to June 2022). PCT levels were measured at admission. Patients' antibiotics use and outcomes were collected via the Society of Critical Care Medicine (SCCM) Viral Infection and Respiratory Illness Universal Study (VIRUS) registry. Patients were stratified into high and low PCT groups based on the first available PCT result. The distinction between high and low PCT was demarcated at both 0.25 ng/ml and 0.50 ng/ml. RESULTS: Our cohort consisted of 3665 patients admitted with COVID-19 pneumonia. The population was predominantly male, Caucasian and non-Hispanic. With the PCT cut-off of 0.25 ng/ml, 2375 (64.8 %) patients had a PCT level <0.25 ng/mL, and 1290 (35.2 %) had PCT ≥0.25 ng/ml. While when the PCT cut off of 0.50 ng/ml was used we observed 2934 (80.05 %) patients with a PCT <0.50 ng/ml while 731(19.94 %) patients had a PCT ≥0.50 ng/ml. Patients with higher PCT levels exhibited significantly higher rates of bacterial infections (0.25 ng/ml cut-off: 4.2 % vs 7.9 %; 0.50 ng/ml cut-off: 4.6 % vs 9.2 %). Antibiotics were used in 66.0 % of the cohort. Regardless of the PCT cutoffs, the antibiotics group showed increased hospital length of stay (LOS), intensive care unit (ICU) admission rate, and mortality. However, early de-escalation (<24 h) of antibiotics correlated with reduced hospital LOS, ICU LOS, and mortality. These results were consistent even after adjusting for confounders. CONCLUSION: Our study shows a substantial number of COVID-19 pneumonia patients received antibiotics despite a low incidence of bacterial infections. Therefore, antibiotics use in COVID pneumonia patients with PCT <0.5 in the absence of clinical evidence of bacterial infection has no beneficial effect.

Hepatocellular carcinoma metastatic to the pituitary gland without an identifiable primary lesion.

Hepatocellular carcinoma is one of the most common malignancies worldwide. However, brain metastases from this cancer are incredibly rare. While the hepatocellular carcinoma mortality rate in the United States has been increasing, hepatocellular carcinoma is rare among patients without underlying liver disease. Here we present a patient with a history of left optic nerve meningioma treated with stereotactic radiosurgery who presented with acute vision loss. Magnetic resonance imaging revealed an enhancing mass lesion in the region of the sella turcica. Neurosurgical histopathology revealed a metastatic lesion consistent with hepatocellular carcinoma. Systemic workup failed to identify a primary liver lesion.

The Year in Cardiothoracic and Vascular Anesthesia: Selected Highlights From 2023.

This special article is the 16th in an annual series for the Journal of Cardiothoracic and Vascular Anesthesia. The authors thank the editor-in-chief, Dr. Kaplan, and the editorial board for the opportunity to continue this series, namely the research highlights of the past year in the specialty of cardiothoracic and vascular anesthesiology. The major themes selected for 2023 are outlined in this introduction, and each highlight is reviewed in detail in the main article. The literature highlights in the specialty for 2023 begin with an update on perioperative rehabilitation in cardiothoracic surgery, with a focus on novel methods to best assess patients in the preoperative and postoperative periods, and the impact of rehabilitation on outcomes. The second major theme is focused on cardiac surgery, with the authors discussing new insights into inhaled pulmonary vasodilators, coronary revascularization surgery, and discussion of causes of coronary graft failure after surgery. The third theme is focused on cardiothoracic transplantation, with discussions focusing on bridge-to-transplantation strategies. The fourth theme is focused on mechanical circulatory support, with discussions focusing on both temporary and durable support. The fifth and final theme is an update on medical cardiology, with a focus on outcomes of invasive approaches to heart disease. The themes selected for this article are only a few of the diverse advances in the specialty during 2023. These highlights will inform the reader of key updates on various topics, leading to improved perioperative outcomes for patients with cardiothoracic and vascular disease.

Spatial and Temporal Mapping of Breast Cancer Lung Metastases Identify TREM2 Macrophages as Regulators of the Metastatic Boundary.

Cancer mortality primarily stems from metastatic recurrence, emphasizing the urgent need for developing effective metastasis-targeted immunotherapies. To better understand the cellular and molecular events shaping metastatic niches, we used a spontaneous breast cancer lung metastasis model to create a single-cell atlas spanning different metastatic stages and regions. We found that premetastatic lungs are infiltrated by inflammatory neutrophils and monocytes, followed by the accumulation of suppressive macrophages with the emergence of metastases. Spatial profiling revealed that metastasis-associated immune cells were present in the metastasis core, with the exception of TREM2+ regulatory macrophages uniquely enriched at the metastatic invasive margin, consistent across both murine models and human patient samples. These regulatory macrophages (Mreg) contribute to the formation of an immune-suppressive niche, cloaking tumor cells from immune surveillance. Our study provides a compendium of immune cell dynamics across metastatic stages and niches, informing the development of metastasis-targeting immunotherapies. SIGNIFICANCE: Temporal and spatial single-cell analysis of metastasis stages revealed new players in modulating immune surveillance and suppression. Our study highlights distinct populations of TREM2 macrophages as modulators of the microenvironment in metastasis, and as the key immune determinant defining metastatic niches, pointing to myeloid checkpoints to improve therapeutic strategies. This article is featured in Selected Articles from This Issue, p. 2489.

Unexpected Candidal Hyphae in Oral Mucosa Lesions-A Clinico-Pathological Study.

Background: Oral mucosal biopsies might harbor candidal hyphae (CH) in the absence of any clinical signs or symptoms. Aim: To assess oral mucosa biopsies for the frequency of unexpected CH and characterize their clinico-pathological features. Materials and Methods: All biopsy reports (2004−2019) were searched using CH/candida/candidiasis as key words. Cases with clinical diagnosis of oral candidiasis (OC) were excluded. Demographic data, health status, smoking habits, clinical features and diagnoses were collected. Statistical analysis included the chi-square test; significance was set at p < 0.05. Results: Of all the biopsies, 100 (1.05%) reported microscopical evidence of CH without typical clinical signs/symptoms of OC. Fifteen cases were from healthy, non-smoking patients. CH was common on buccal mucosa (38%) and lateral tongue (23%). The tip of tongue (OR = 54.5, 95% CI 9.02−329.4, p < 0.001) and lateral tongue (OR = 3.83, 95% CI 2.4−6.09, p < 0.001) were more likely to harbor CH-positive lesions. CH-positive lesions were diagnosed as epithelial hyperplasia (55%) and exophytic reactive lesions (30%). No correlation was found between CH and the grade of epithelial dysplasia. Conclusions: Microscopic evidence of CH embedded into oral epithelium without typical signs/symptoms of OC is rare, especially in healthy, non-smokers. Since CH was occasionally found in oral sites prone to local trauma and in association with reactive lesions, in absence of host co-morbidities, the contribution of local mechanical forces to CH embedment cannot be ruled out.

The interaction of CD4+ helper T cells with dendritic cells shapes the tumor microenvironment and immune checkpoint blockade response.

Despite their key regulatory role and therapeutic potency, the molecular signatures of interactions between T cells and antigen-presenting myeloid cells within the tumor microenvironment remain poorly characterized. Here, we systematically characterize these interactions using RNA sequencing of physically interacting cells (PIC-seq) and find that CD4+PD-1+CXCL13+ T cells are a major interacting hub with antigen-presenting cells in the tumor microenvironment of human non-small cell lung carcinoma. We define this clonally expanded, tumor-specific and conserved T-cell subset as T-helper tumor (Tht) cells. Reconstitution of Tht cells in vitro and in an ovalbumin-specific αß TCR CD4+ T-cell mouse model, shows that the Tht program is primed in tumor-draining lymph nodes by dendritic cells presenting tumor antigens, and that their function is important for harnessing the antitumor response of anti-PD-1 treatment. Our molecular and functional findings support the modulation of Tht-dendritic cell interaction checkpoints as a major interventional strategy in immunotherapy.

Relationship between religiosity, spirituality and physical and mental outcomes in fibromyalgia patients.

OBJECTIVES: The coping mechanisms utilised by patients with the fibromyalgia syndrome (FM) pose a crucial focus of treatment. Previous research points to the positive effects of religiosity and spirituality (R/S) as tools for coping with illness. The role of these factors in coping with chronic pain in FM has not previously been studied. The aim of this study was to evaluate the link between R/S and FM outcomes. METHODS: Fifty-five FM patients (ACR criteria) attending a tertiary rheumatology clinic completed a packet of questionnaires assessing demographic data, levels of religiosity and spirituality (SpREUK) and locus of control (LOC). These variables were then individually assessed for influence on FM outcome measures, using the Fibromyalgia Impact Questionnaire (FIQ), the SF-36, and the Beck Depression Index (BDI). RESULTS: A high score on SpREUK I (search for meaningful support) was negatively correlated with the Role-Physical (p=0.032) and Role-Emotional (p<0.005) scales on SF-36. Secular patients scored higher on SF-36 domains of "Role limitation due to emotional health" and "General health" (p<0.05). Employment demonstrated a positive correlation with the FIQ (p<0.01), the BDI (p<0.001), and the SF-36 (p<0.05). Physical activity correlated positively with BDI scores (p=0.012) and better scores on SF-36: energy/fatigue (p=0.024), social-functioning (p=0.014) and physical-functioning (p<0.01). No significant correlation was found between LOC (internal versus external) and FM outcomes. No significant correlation was found between SpREUK domains and the BDI. CONCLUSIONS: FM patients do not appear to benefit from high levels of R/S. Physicians should be aware of the impact of R/S on well-being in this population.

Anticoagulant and antiplatelet treatment in cancer patients with thrombocytopenia.

A B S T R A C T Antithrombotic therapy (anticoagulation or antiplatelet therapy) is frequently prescribed in cancer patients for prior or new indications such as venous thromboembolism, secondary prevention of arterial thrombosis or atrial fibrillation. Therefore, it is not uncommon for thrombocytopenic cancer patients to have an indication for antithrombotic therapy. Thrombocytopenia does not reduce the risk of recurrent thrombosis. The bleeding risk with anticoagulation appears to increase when platelets are <50×109/L, but individual platelet counts are poor predictors of bleeding. Management options when platelets are <50×109/L include no change, temporarily withholding antithrombotic therapy, reducing dose, changing the regimen, and increasing the platelet transfusion threshold. There are currently no data on use of direct oral anticoagulants when platelets are below 50×109/L, and there is reason in restricting their use. Little is known on antiplatelet therapy in this setting, although recent data suggest the prognostic importance and apparent safety of aspirin in acute myocardial infarction and thrombocytopenia. This paper will review the evidence, guidelines, current practice and ongoing studies on anticoagulation and antiplatelet therapy in thrombocytopenic patients with cancer.

Massive Pulmonary Hemorrhage from Bronchial Varix.

Bronchial varix is a rare pulmonary disorder which may lead to life-threatening hemorrhage. Diagnosis is difficult because of the subtle abnormalities on radiographic and bronchoscopic examination. We present a case of massive hemoptysis from a bleeding bronchial varix. In the absence of immediate complex endobronchial therapy in the island of Guam, this case was initially managed with nebulized and intravenous tranexamic acid. This was followed by endobronchial blockade of the bleeding airway with endobronchial epinephrine instillation. Selective bronchial artery embolization alleviated the acute-phase bleeding. Prone positioning was initiated due to severe hypoxia after blood clots compromised the patency of bilateral bronchial airways. Prone ventilation was employed for 17 hours for 2 consecutive days with intermittent bronchoscopic forceps extraction of airway blood clots while in the prone position. These maneuvers resulted to improved lung ventilation and oxygenation. The patient underwent bronchial sleeve resection surgery for definitive management.

Anticoagulation in thrombocytopenic patients with hematological malignancy: A multinational clinical vignette-based experiment.

BACKGROUND: Thrombocytopenia in cancer patients with an indication for anticoagulation poses a unique clinical challenge. There are guidelines for the setting of venous thromboembolism but not atrial fibrillation (AF). Evidence is lacking and current practice is unclear. OBJECTIVE: To identify patient and physician characteristics associated with anticoagulation management in hematological malignancy and thrombocytopenia. METHODS: A clinical vignette-based experiment was designed. Eleven hematologists were interviewed, identifying 5 relevant variable categories with 2-5 options each. Thirty hypothetical vignettes were generated. Each physician received 5 vignettes and selected a management strategy (hold anticoagulation; no change; transfuse platelets; modify type/dose). The survey was distributed to hematologists and thrombosis specialists in 3 countries. Poisson regression models with cluster robust variance estimates were used to calculate relative risks for using one management option over the other, for each variable in comparison to a reference variable. RESULTS: 168 physicians answered 774 cases and reported continuing anticoagulation for venous thromboembolism or AF in 607 (78%) cases, usually with dose reduction or platelet transfusion support. Overall, management was affected by platelet count, anticoagulation indication, time since indication, type of hematological disease and treatment, and prior major bleeding, as well as physician demographics and practice setting. The CHA2DS2-VASc score and time since AF diagnosis affected anticoagulation management in AF. CONCLUSION: This study indicates what the widely accepted management strategies are. These strategies, and possibly others, should be assessed prospectively to ascertain effectiveness. The decision process is intricate and compatible with current venous thromboembolism guidelines.

Cancer-associated fibroblasts in the tumor microenvironment of tongue carcinoma is a heterogeneous cell population.

OBJECTIVES: To examine different immunophenotypes of cancer-associated fibroblasts (CAFs) in tongue squamous cell carcinoma (TSCC) and to investigate how they related to clinical outcomes. METHODS: Serial sections from 54 cases of TSCC were immunohistochemically stained with α-smooth muscle actin (αSMA, CAF marker) to determine CAF density, and double-immunostained with αSMA combined with CD80 and CD86 (myeloid/monocytic-derived cell markers), Nanog (mesenchymal stem cell marker) and CD133 (hematopoietic/endothelial stem cell marker). Density of cells co-expressing these marker combinations was semi-quantitatively assessed in 5 randomly selected high power fields within the tumor area and scored as 1 - one-to-five stained cells in each field, 2 - more than 5 stained cells in each field; any finding less than score 1, was allocated a score of 0. RESULTS: There were 26 CAF-poor, 16 CAF-rich and 12 CAF-intermediated cases. CD86+αSMA+ cells were the most frequent (80.4%) followed by CD80+αSMA+ (72%) and Nanog+αSMA+ cells (56%). The CD133+αSMA+ phenotype was found only in association with blood vessels. High density of αSMA+ CAFs was associated with disease recurrence and poor survival (p < 0.05). Increased density of CD86+αSMA+ cells was significantly associated with CAF-rich tumors and with poor survival (p < 0.05). CONCLUSION: In TSCC, CAFs demonstrate heterogeneous and overlapping phenotypes with the myeloid/monocytic type being the most frequent and having an impact on the clinical outcomes. Further studies are needed in order to further characterize CAF phenotypes in carcinomas of various oral sites, as this may open new frontiers for personalized medicine.

Metastatic tumors in oral mucosa and jawbones: Unusual primary origins and unusual oral locations.

AIM: To perform clinico-pathological characterization of a large series of oral metastases, collected from 3 main medical centers in Israel and compare findings to data on frequency of primary cancer types in the population. MATERIALS: Pathology archives were searched for cases of metastatic tumors to the oral soft tissues and jawbones, 1990 - 2016. Metastases to the skin of face or to major salivary glands have been excluded. Demographic data and histopathological features were analyzed. RESULTS: Study population included 60 patients, 35 females and 25 males (ratio of 1.4:1). The age range was 17-87 years, mean 67.7 + 14.36 years. Only 3 (5%) patients were under 40 years, the remaining clustered predominantly in the 60-80 year age group. The mean age of females (59 + 13.84) was significantly lower than that of males (67.44 + 14) (p = 0.03). There was an almost equal distribution between the oral soft tissue and the jawbones (48.3% and 51.7%, respectively). The five most common organs from which metastases were distributed to the oral cavity and jawbones combined were kidney (20%), breast (15%), cutaneous (predominately melanoma, 13%), lung (11.7%) and soft tissue-sarcomas (8.3%). For comparison, Israel National Cancer Registry 2013 reported that the most frequent malignancies were breast (25.8%), colorectal cancer (16.3%), lung (12%) and prostate (10%). Malignant melanoma was 6th (5.4%), kidney malignancy was only 9th in frequency (4.2%). Although the gingiva and jawbones were the most frequent locations, some cases presented in unusual locations, (mandibular vestibule, lower lip, posterior dorsal tongue), without any specific clinical feature to suggest metastasis. CONCLUSIONS: The most frequent primary origins for oral metastasis do not correspond to the relative frequency of the primary tumors in the population, indicating that metastatic spread is not a random process. Although the majority of metastasis involves the gingiva and jawbones, any other oral mucosal location might be involved. Thus, in adult/older patients, metastasis from a distant site should be included in the differential diagnosis of oral masses at any oral location, whether the existence of a primary tumor is reported or not.

Markers of the pre-metastatic niche "knock on the door" of metastasis-free cervical lymph nodes in patients with oral cancer.

AIM: To assess expression of some markers of the pre-metastatic niche (PMN) in lymph nodes (LNs) of oral cancer patients. MATERIALS: LNs from metastatic-free neck dissections (LN0/N0, N = 43) and metastatic-free LNs in the vicinity of metastasis-containing LNs (LN0/N+, N = 30) were immuno-histochemically stained for lysyl oxidase (LOX), fibronectin (FN), vascular-endothelial growth factor receptor (VEGFR)-1 and matrix metalloproteinase (MMP)-9. Staining was assessed as 0 (no or weak staining), 1 (strong stain in 25% cells or extracellular area), 2 (same as 1 but in up to 50%) and 3 (same as 1 but in > than 50% of cells/area). Assessment was performed in the lymph node capsule (CAP), sub-capsular sinus (SCS) and medullary sinus (MS). In addition, sections were stained with picrosirius red and examined with polarized microscopy for assessing the distribution of polarization colors of the collagen fibers in the LN capsular area. RESULTS: All examined LNs were positive for markers of the PMN. In general, the distribution and intensity of the immunoreactivity was similar between the LN0/N0 and LN0/N+, with only a few differences regarding expression of LOX in the capsule (p = 0.002) and VEGFR1 and MMP9 in the SCS (p = 0.023 and p < 0.001, respectively). Picrosirius red stain and polarized microscopy revealed a disrupted arrangement and distribution of the collagen fibers in both LN0/N0 and LN0/N + . CONCLUSION: Markers for PMN were shown for the first time to be expressed in cervical LN0/N0 from patients with oral cancer, suggesting the increased permissive pathway remotely paved by the primary oral tumor for the incoming metastatic cells.

Current treatment of lymphoma in pregnancy.

Introduction: Lymphoproliferative diseases occurring during pregnancy present the treating physician with unique diagnostic and therapeutic challenges, aiming to achieve maternal cure without impairing fetal health, growth, and survival. Due to the rarity of this complication, there is limited data to guide clinical decision-making, especially regarding the safety of novel emerging therapies. Areas covered: The presented review describes the current practice of treatment for Hodgkin's (HL) and non-Hodgkin's (NHL) lymphoma in the pregnant patient, according to disease stage and trimester of pregnancy. Novel agents for treatment of lymphoma in the setting of pregnancy are discussed. Therapeutic dilemmas and areas of uncertainty are illuminated. Expert opinion: HL and NHL are potentially curable diseases in the pregnant patient with generally good outcomes for the mother and the offspring, when tailoring the treatment according to the individual patient. The complexity of the situation merits shared decision-making with the patient and her family, explicitly outlining the risks and benefits. The pregnant patient is best managed by a multidisciplinary team, familiar with the intricacies of the gestational period, and providing the necessary support and sensitivity. Further studies are needed regarding the safety of novel agents in pregnancy.

An in-silico quality assurance study of contouring target volumes in thoracic tumors within a cooperative group setting.

INTRODUCTION: Target delineation variability is a significant technical impediment in multi-institutional trials which employ intensity modulated radiotherapy (IMRT), as there is a real potential for clinically meaningful variances that can impact the outcomes in clinical trials. The goal of this study is to determine the variability of target delineation among participants from different institutions as part of Southwest Oncology Group (SWOG) Radiotherapy Committee's multi-institutional in-silico quality assurance study in patients with Pancoast tumors as a "dry run" for trial implementation. METHODS: CT simulation scans were acquired from four patients with Pancoast tumor. Two patients had simulation 4D-CT and FDG-FDG PET-CT while two patients had 3D-CT and FDG-FDG PET-CT. Seventeen SWOG-affiliated physicians independently delineated target volumes defined as gross primary and nodal tumor volumes (GTV_P & GTV_N), clinical target volume (CTV), and planning target volume (PTV).Six board-certified thoracic radiation oncologists were designated as the 'Experts' for this study. Their delineations were used to create a simultaneous truth and performance level estimation (STAPLE) contours using ADMIRE software (Elekta AB, Sweden 2017). Individual participants' contours were then compared with Experts' STAPLE contours. RESULTS: When compared to the Experts' STAPLE, GTV_P had the best agreement among all participants, while GTV_N showed the lowest agreement among all participants. There were no statistically significant differences in all studied parameters for all TVs for cases with 4D-CT versus cases with 3D-CT simulation scans. CONCLUSIONS: High degree of inter-observer variation was noted for all target volume except for GTV_P, unveiling potentials for protocol modification for subsequent clinically meaningful improvement in target definition. Various similarity indices exist that can be used to guide multi-institutional radiotherapy delineation QA credentialing.

Intravenous immunoglobulin-induced acute thrombocytopenia.

BACKGROUND: Intravenous immunoglobulin (IVIG) has known efficacy in various hematologic conditions, including immune thrombocytopenic purpura. STUDY DESIGN AND METHODS: We present the clinical course of a patient with splenic marginal zone lymphoma, who developed acute thrombocytopenia on three consecutive episodes, with nadir counts of 27 × 109 , 50 × 109 , and 9 × 109 /L, upon administration of Intratect IVIG for hypogammaglobulinemia. An immunofluorescence test applying flow cytometry and monoclonal antibody immobilization of platelet antigens (MAIPA) assay were used to evaluate the reaction between IgG present in the IVIG preparations and the patient's or healthy donors' platelets (PLTs). RESULTS: A strong direct binding reaction was observed between the patient's PLTs and Intratect IgG using both methods. A similar reaction failed to materialize with controls. Binding was not antigen specific according to MAIPA. CONCLUSIONS: This is the first reported case of thrombocytopenia as a possible adverse effect of IVIG.

Extended Small-Dose Platelet Transfusions in Multitransfused Hemato-Oncological Patients: A Single-Center Experience.

BACKGROUND: Refractoriness to platelet transfusion, prevalent among 15-20% of hemato-oncological patients, is associated with multitransfusions and inferior outcomes. We evaluated the effectiveness of extended slow-dose transfusion (ESDT) in increasing platelet increments in multitransfused patients. METHODS: Patients treated after the implementation of ESDT were compared with historical controls treated with standard single-donor platelet (SDP) transfusions. Cohorts of early and late recipients were assembled for comparison, i.e. the 8th or 9th and 11th platelet unit per patient, respectively. Patients in the ESDT group received transfusions equal to half an SDP unit, administered over 4 h. Effectiveness was defined as a higher corrected count increment (CCI) at 1, 12, and 24 h after transfusion. RESULTS: In the early-recipients cohort, 24-h-posttransfusion increments were available for 29 ESDT patients and 6 standard patients, and did not differ significantly between the groups (p = 0.078). The 24-h-posttransfusion increment was available for 20 ESDT patients and 7 standard patients in the late-recipients cohort. The CCI was significantly higher in the ESDT group (p = 0.042). ABO compatibility improved the CCI (p = 0.01). CONCLUSIONS: ESDT demonstrated slightly higher increments at 24 h after transfusion in late recipients, suggesting this could be a cost-effective approach for the treatment of thrombocytopenic multitransfused hemato-oncological patients.

Protein Carbonylation of an Amino Acid Residue of the Na/K-ATPase α1 Subunit Determines Na/K-ATPase Signaling and Sodium Transport in Renal Proximal Tubular Cells.

BACKGROUND: We have demonstrated that cardiotonic steroids, such as ouabain, signaling through the Na/K-ATPase, regulate sodium reabsorption in the renal proximal tubule. By direct carbonylation modification of the Pro222 residue in the actuator (A) domain of pig Na/K-ATPase α1 subunit, reactive oxygen species are required for ouabain-stimulated Na/K-ATPase/c-Src signaling and subsequent regulation of active transepithelial (22)Na(+) transport. In the present study we sought to determine the functional role of Pro222 carbonylation in Na/K-ATPase signaling and sodium handling. METHODS AND RESULTS: Stable pig α1 knockdown LLC-PK1-originated PY-17 cells were rescued by expressing wild-type rat α1 and rat α1 with a single mutation of Pro224 (corresponding to pig Pro222) to Ala. This mutation does not affect ouabain-induced inhibition of Na/K-ATPase activity, but abolishes the effects of ouabain on Na/K-ATPase/c-Src signaling, protein carbonylation, Na/K-ATPase endocytosis, and active transepithelial (22)Na(+) transport. CONCLUSIONS: Direct carbonylation modification of Pro224 in the rat α1 subunit determines ouabain-mediated Na/K-ATPase signal transduction and subsequent regulation of renal proximal tubule sodium transport.