A Case of Hypernatremia in a Newly Diagnosed Patient With Acute Myeloid Leukemia: Lessons for Nephrologists.

Arginine vasopressin deficiency (AVP-D), formerly known as central diabetes insipidus, is a disease characterized by polyuria, polydipsia, and hypernatremia. The concomitant diagnosis of acute myeloid leukemia (AML) is an underappreciated event that requires prompt recognition and treatment by practicing nephrologists and hematologists. This report highlights this importance by describing the case of a 39-year-old patient newly diagnosed with AML who developed severe hypernatremia. The role of diagnostic testing through desmopressin (DDAVP) challenge and copeptin testing to confirm the diagnosis of AVP-D in this context and the use of DDVAP for treatment are discussed. Practicing nephrologists and primary care providers taking care of patients with similar symptoms will benefit from understanding the pathophysiology of AVP-D, its relationship with AML, and the prognosis in this patient cohort.

Homodimeric Granzyme A Opsonizes Mycobacterium tuberculosis and Inhibits Its Intracellular Growth in Human Monocytes via Toll-Like Receptor 4 and CD14.

Mycobacterium tuberculosis (Mtb)-specific γ9δ2 T cells secrete granzyme A (GzmA) protective against intracellular Mtb growth. However, GzmA-enzymatic activity is unnecessary for pathogen inhibition, and the mechanisms of GzmA-mediated protection remain unknown. We show that GzmA homodimerization is essential for opsonization of mycobacteria, altered uptake into human monocytes, and subsequent pathogen clearance within the phagolysosome. Although monomeric and homodimeric GzmA bind mycobacteria, only homodimers also bind cluster of differentiation 14 (CD14) and Toll-like receptor 4 (TLR4). Without access to surface-expressed CD14 and TLR4, GzmA fails to inhibit intracellular Mtb. Upregulation of Rab11FIP1 was associated with inhibitory activity. Furthermore, GzmA colocalized with and was regulated by protein disulfide isomerase AI (PDIA1), which cleaves GzmA homodimers into monomers and prevents Mtb inhibitory activity. These studies identify a previously unrecognized role for homodimeric GzmA structure in opsonization, phagocytosis, and elimination of Mtb in human monocytes, and they highlight PDIA1 as a potential host-directed therapy for prevention and treatment of tuberculosis, a major human disease.

Inhaled medications for chronic obstructive pulmonary disease predict surgical complications and survival in stage I non-small cell lung cancer.

Background: Lung function is routinely assessed prior to surgical resection for non-small cell lung cancer (NSCLC). Further assessment of chronic obstructive pulmonary disease (COPD) using inhaled COPD medications to determine disease severity, a readily available metric of disease burden, may predict postoperative outcomes and overall survival (OS) in lung cancer patients undergoing surgery. Methods: We retrospectively evaluated clinical stage I NSCLC patients receiving surgical treatment within the Veterans Health Administration from 2006-2016 to determine the relationship between number and type of inhaled COPD medications (short- and long-acting beta2-agonists, muscarinic antagonists, or corticosteroids prescribed within 1 year before surgery) and postoperative outcomes including OS using multivariable models. We also assessed the relationship between inhaled COPD medications, disease severity [measured by forced expiratory volume in 1 second (FEV1)], and diagnosis of COPD. Results: Among 9,741 veterans undergoing surgery for clinical stage I NSCLC, patients with COPD were more likely to be prescribed inhaled medications than those without COPD [odds ratio (OR) =5.367, 95% confidence interval (CI): 4.886-5.896]. Increased severity of COPD was associated with increased number of prescribed inhaled COPD medications (P<0.0001). The number of inhaled COPD medications was associated with prolonged hospital stay [adjusted OR (aOR) =1.119, 95% CI: 1.076-1.165), more major complications (aOR =1.117, 95% CI: 1.074-1.163), increased 90-day mortality (aOR =1.088, 95% CI: 1.013-1.170), and decreased OS [adjusted hazard ratio (aHR) =1.061, 95% CI: 1.042-1.080]. In patients with FEV1 ≥80% predicted, greater number of prescribed inhaled COPD medications was associated with increased 30-day mortality (aOR =1.265, 95% CI: 1.062-1.505), prolonged hospital stay (aOR =1.130, 95% CI: 1.051-1.216), more major complications (aOR =1.147, 95% CI: 1.064-1.235), and decreased OS (aHR =1.058, 95% CI: 1.022-1.095). When adjusting for other drug classes and covariables, short-acting beta2-agonists were associated with increased 90-day mortality (aOR =1.527, 95% CI: 1.120-2.083) and decreased OS (aHR =1.087, 95% CI: 1.005-1.177). Conclusions: In patients with early-stage NSCLC, inhaled COPD medications prescribed prior to surgery were associated with both short- and long-term outcomes, including in patients with FEV1 ≥80% predicted. Routine assessment of COPD medications may be a simple method to quantify operative risk in early-stage NSCLC patients.

Improved Purification of Human Granzyme A/B and Granulysin Using a Mammalian Expression System.

Cytotoxic lymphocytes release proteins contained within the cytoplasmic cytolytic granules after recognition of infected or tumor target cells. These cytotoxic granular proteins (namely granzymes, granulysin, and perforin) are key immunological mediators within human cellular immunity. The availability of highly purified cytotoxic proteins has been fundamental for understanding their function in immunity and mechanistic involvement in sepsis and autoimmunity. Methods for recovery of native cytotoxic proteins can be problematic leading to: 1) the co-purification of additional proteins, confounding interpretation of function, and 2) low yields of highly purified proteins. Recombinant protein expression of individual cytolytic components can overcome these challenges. The use of mammalian expression systems is preferred for optimal post-translational modifications and avoidance of endotoxin contamination. Some of these proteins have been proposed for host directed human therapies (e.g. - granzyme A), or treatment of systemic infections or tumors as in granulysin. We report here a novel expression system using HEK293T cells for cost-effective purification of high yields of human granzymes (granzyme A and granzyme B) and granulysin with enhanced biological activity than previous reports. The resulting proteins are free of native contaminants, fold correctly, and remain enzymatically active. Importantly, these improvements have also led to the first purification of biologically active recombinant human granulysin in high yields from a mammalian system. This method can be used as a template for purification of many other secreted cellular proteins and may lead to advances for human medicine.

Identification of novel neutrophil very long chain plasmalogen molecular species and their myeloperoxidase mediated oxidation products in human sepsis.

Plasmalogens are a class of phospholipids containing vinyl ether linked aliphatic groups at the sn-1 position. Plasmalogens are known to contain 16- and 18-carbon aliphatic groups at the sn-1 position. Here, we reveal that the human neutrophil plasmenylethanolamine pool uniquely includes molecular species with very long carbon chain (VLC) aliphatic groups, including 20-, 22- and 24-carbon vinyl ether linked aliphatic groups at the sn-1 position. We identified these novel VLC plasmalogen species by electrospray ionization mass spectrometry methods. VLC plasmalogens were only found in the neutrophil plasmenylethanolamine pool. During neutrophil activation, VLC plasmenylethanolamines undergo myeloperoxidase-dependent oxidation to produce VLC 2-chlorofatty aldehyde and its oxidation product, 2-chlorofatty acid (2-ClFA). Furthermore, plasma concentrations of VLC 2-ClFA are elevated in human sepsis. These studies demonstrate for the first time VLC plasmenylethanolamine molecular species, their myeloperoxidase-mediated chlorolipid products and the presence of these chlorolipids in human sepsis.

Granzyme A Produced by γ9δ2 T Cells Activates ER Stress Responses and ATP Production, and Protects Against Intracellular Mycobacterial Replication Independent of Enzymatic Activity.

Mycobacterium tuberculosis (Mtb), the pathological agent that causes tuberculosis (TB) is the number one infectious killer worldwide with one fourth of the world's population currently infected. Data indicate that γ9δ2 T cells secrete Granzyme A (GzmA) in the extracellular space triggering the infected monocyte to inhibit growth of intracellular mycobacteria. Accordingly, deletion of GZMA from γ9δ2 T cells reverses their inhibitory capacity. Through mechanistic studies, GzmA's action was investigated in monocytes from human PBMCs. The use of recombinant human GzmA expressed in a mammalian system induced inhibition of intracellular mycobacteria to the same degree as previous human native protein findings. Our data indicate that: 1) GzmA is internalized within mycobacteria-infected cells, suggesting that GzmA uptake could prevent infection and 2) that the active site is not required to inhibit intracellular replication. Global proteomic analysis demonstrated that the ER stress response and ATP producing proteins were upregulated after GzmA treatment, and these proteins abundancies were confirmed by examining their expression in an independent set of patient samples. Our data suggest that immunotherapeutic host interventions of these pathways may contribute to better control of the current TB epidemic.

Structural and catalytic effects of proline substitution and surface loop deletion in the extended active site of human carbonic anhydrase II.

UNLABELLED: Bioengineering of a thermophilic enzyme starting from a mesophilic scaffold has proven to be a significant challenge, as several stabilizing elements have been proposed to be the foundation of thermal stability, including disulfide bridges, surface loop reduction, ionic pair networks, proline substitutions and aromatic clusters. This study emphasizes the effect of increasing the rigidity of human carbonic anhydrase II (HCA II; EC 4.2.1.1) via incorporation of proline residues at positions 170 and 234, which are located in surface loops that are able to accommodate restrictive main-chain conformations without rearrangement of the surrounding peptide backbone. Additionally, the effect of the compactness of HCA II was examined by deletion of a surface loop (residues 230-240) that had been previously identified as a possible source of thermal stability for the hyperthermophilic carbonic anhydrase isolated from the bacterium Sulfurihydrogenibium yellowstonense YO3AOP1. Differential scanning calorimetry analysis of these HCA II variants revealed that these structural modifications had a minimum effect on the thermal stability of the enzyme, while kinetic studies showed unexpected effects on the catalytic efficiency and proton transfer rates. X-ray crystallographic analysis of these HCA II variants showed that the electrostatic potential and configuration of the highly acidic loop (residues 230-240) play an important role in its high catalytic activity. Based on these observations and previous studies, a picture is emerging of the various components within the general structural architecture of HCA II that are key to stability. These elements may provide blueprints for rational thermal stability engineering of other enzymes. DATABASE: Structural data have been submitted to the Protein Data Bank under accession numbers 4QK1 (K170P), 4QK2 (E234P) and 4QK3 (Δ230-240).