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.
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 γ
9
δ
2
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 γ
9
δ
2
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.
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.
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.
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•Novel very long chain (VLC) plasmenylethanolamine molecular species are discovered in human neutrophils.•VLC plasmenylethanolamines undergo myeloperoxidase dependent oxidation to produce new VLC chlorinated lipids.•VLC 2-chlorofatty acids are elevated in human sepsis.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Abstract
γδ T cells are ideal effector cells against cancer and pathogens for numerous reasons: they are found in blood, mucosa, and lymphoid organs, develop memory responses independent of MHC ...restriction, present antigen, produce cytokines, and are cytolytic. An obstacle for the development of biomedical interventions targeting γδ T cells is the need for an adjuvant that promotes their expansion and is suitable for human use. The adjuvant routinely used, IL-2, has multiple drawbacks limiting its use for vaccination. Our lab has shown that memory γ 9δ 2T cells, induced by BCG vaccination, are expanded in vitro by M. tuberculosis, M.bovis(BCG), and our lab’s vaccine candidate 6-O-methylglucoselipopolysaccharide (mGLP), in the presence of IL-2. Additionally, the expanded γ 9δ 2T cells inhibit intracellular mycobacterial growth. This inhibition is dependent on granzyme A (GzmA) production by γ 9δ 2T cells, and GzmA alone can inhibit intracellular mycobacterial growth.
We recently identified that GzmA can also promote expansion of γδ T cells, without exogenous IL-2, in response to mGLP and HMBPP, a phosphoantigen that stimulates all γ 9δ 2T cells. To identify γδ T cell adjuvants, in vitro expansion assays were performed using human peripheral blood mononuclear cells, dendritic cells, antigen, and adjuvant. The absolute numbers of expanded effector γδ T cells were calculated using flow cytometry after 7 days of culture. We identified that the ability of GzmA to promote γδ T cell expansion is dependent upon TLR4, CD14, and GzmA homodimerization, but independent of GzmA enzymatic activity. This new information has broad implications for vaccine design, promoting γδ T cell responses, and understanding the role of GzmA in infectious disease and cancer.
Supported by grants from NIH (R01 AI048391) and Bill & Melinda Gates Foundation (OP1118659)
Abstract
γδ T cells are ideal effector cells against infectious diseases and cancer. They reside in blood, lymphoid organs, and mucosa, readily available for host defense. γδ T cells develop memory ...responses independent of MHC presentation. They perform immune functions such as cytolysis, cytokine production, antigen presentation, dendritic cell maturation, and promotion of B cell and T cell responses. Our lab reported that γδ T cells responsive to M. tuberculosis-specific 6-O-methylglucose-containing lipopolysaccharides (mGLP) inhibit intracellular mycobacterial growth. Preliminary data from NHP studies indicate that vaccination with mGLP and IL-2 greatly reduces Mtb burden post-challenge. IL-2 is not translatable for adjuvant use in humans. Therefore, novel adjuvants for γδ T cells are needed to develop optimal vaccine strategies. We are investigating adjuvant candidates to determine their ability to induce optimal activation and expansion of γδ T cells with mGLP or the phosphoantigen HMBPP. To screen adjuvants, PBMCs are incubated with antigen and adjuvant for 7 days, then analyzed for expansion of γδ T cells. Initially few adjuvants promoted γδ T cell expansion in response to HMBPP and none to mGLP. When matured monocyte-derived DCs were added, multiple adjuvants promoted γδ T cell expansion in response to HMBPP and mGLP stimulation without IL-2. We will determine mechanisms involved in novel adjuvant activity, whether adjuvant and mGLP-expanded γδ T cells inhibit intracellular mycobacterial growth, identify cytokines involved in candidate adjuvant effects, characterize cell surface markers upregulated on DC and γδ T cells, and test the best candidate adjuvant(s) with mGLP in NHP trials.
Supported by Gama Delta R01 AI048391
Abstract
One fourth of the world population is infected with Tuberculosis (TB). Our lab has identified γ9δ2 T cells that secrete Granzyme A (GzmA) with TB protective effects. In this study, we ...investigated the mechanism(s) by which human GzmA inhibits the intracellular replication of mycobacteria within infected human primary monocytes.
GzmA was added to mycobacteria-infected monocytes for downstream analyses using 2D-DIGE and shotgun proteomics. We generated WT, enzymatically inactive (S195A), and monomeric only (C93S) recombinant GzmA and performed: flow cytometry studies using viable mycobacteria; intracellular inhibition assays neutralizing CD14, TLR4 and TLR2; and GzmA immunoprecipitation experiments.
The 2D-DIGE proteomic analyses found the ER-stress response and ATP metabolism pathways as important for GzmA-mediated inhibition. Separately, shotgun proteomics uncovered the upregulation of Rab11FIP1 (important for phagocytosis). Both GzmA-WT and S195A proteins inhibited intracellular mycobacteria, but C93S did not. Neutralization of CD14 and TLR4, but not TLR2, reversed GzmA-inhibitory activity. GzmA-WT, S195A, and C93S all bound mycobacteria. However, GzmA-WT and S195A, but not GzmA-C93S, stably bound to TLR4 and CD14.
Collectively, these studies demonstrate key structural, functional, and inter-/intra-molecular features required for GzmA-mediated inhibition of intracellular mycobacteria including interactions between GzmA, mycobacteria, TLR4 and CD14. These interactions result in the ER stress response, altered ATP metabolism, enhanced phagocytosis, and inhibition of mycobacteria. Thus, GzmA’s potential role as opsonin could lead to novel host-directed therapeutics for TB infections.
Supported by grants from NIH (F30HL151136-01, R01AI048391-12)
One fourth of the world population is infected with Mycobacterium tuberculosis (Mtb), the pathogen that causes Tuberculosis (TB). Our lab has identified γ9δ2 T cells that secrete Granzyme A (GzmA) ...with TB protective effects as a key mediator of host-pathogen interactions. GzmA is a serine protease found as homodimers within granules of cytotoxic T lymphocytes. In this dissertation, the mechanism by which GzmA inhibits the intracellular replication of mycobacteria within infected human primary monocytes was investigated. Human GzmA was added to mycobacteria-infected primary monocytes from multiple volunteers, and their cell lysates were labeled and analyzed using 2D-DIGE as well as shotgun global proteomics. GzmA was expressed in a mammalian expression system and included wild type (WT) protein, or enzymatically inactive (S195A) and monomeric (C93S) only mutated variants. The Mycobacterial Growth Inhibition Assays (MGIA) were performed to investigate whether GzmA protease activity or homodimeric structure are necessary. Flow cytometry experiments were conducted to analyze whether GzmA-WT, S195A, C93S could stably bind to mycobacteria. Antibody neutralization experiments were conducted with antibodies against CD14, TLR4 and TLR2. Immunoprecipitation experiments were performed to understand if GzmA stably binds to CD14 and TLR4. Finally, confocal microscopy experiments were performed to investigate the GzmA-mediated inhibition kinetics. The proteomic approach identified two pathways, the ER-stress response and ATP metabolism as important for GzmA-mediated inhibition of intracellular mycobacteria. Separately, shotgun proteomics uncovered the upregulation of Rab11FIP1 protein, which is an important factor for effective phagocytosis, highlighting that GzmA may be acting as an opsonin to facilitate mycobacterial uptake and degradation. Both GzmA-WT and S195A proteins can inhibit intracellular mycobacteria, but C93S cannot. Neutralization of CD14 and TLR4, but not TLR2, reverse GzmA-inhibitory activity. GzmA-WT, S195A, and C93S all bind mycobacteria. However, GzmA-WT and S195A, but not GzmA-C93S, stably bind to TLR4 and CD14. Confocal experiments showed that GzmA is internalized inside monocytes and within 3 hours mycobacteria were cleared from the infected cells.Since both enzymatically active and inactive GzmA are able to inhibit intracellular mycobacteria, but monomeric GzmA-C93S does not, GzmA protein structure is a key determinant for: 1) binding to mycobacteria, 2) potentiating pathogen phagocytosis, 3) enhancing activation of monocytes involving CD14 and TLR4, and 4) activating ER stress response and ATP production to neutralize intracellular pathogen. These studies have led to a better understanding of GzmA biology during infection and will facilitate development of novel host-directed therapeutics for control of Mtb infection.
Abstract
One fourth of the world population is infected with Mycobacterium tuberculosis (Mtb), the pathogen that causes Tuberculosis (TB). Our lab has identified γδ T cells that secrete Granzyme A ...(GzmA) with TB protective effects. To elucidate the mechanism(s) involved in GzmA-mediated mycobacterial pathogen inhibition using a relevant in vitro human system. This GzmA was utilized to treat mycobacteria-infected primary monocytes from multiple volunteers, and their cell lysates were analyzed using 2D-DIGE global proteomics. A catalytically inactive GzmA variant was produced to study the enzymatic role in the Mycobacterial Growth Inhibition Assay (MGIA). In parallel, confocal microscopy experiments looked at GzmA internalization. We have evidence that Granzyme A’s intact active site is not necessary to inhibit the intracellular replication of mycobacteria as the S195A variant inhibits mycobacterial infection in MGIA as Wild Type (WT) GzmA. The proteomic approach identified two pathways: ER-stress response and purinergic channel receptor activation are important for GzmA-mediated intracellular mycobacterial inhibition. These results were validated in separate volunteers by western blot. Confocal microscopy experiments show that GzmA is internalized within the infected cell and clears the infection. Modulation of key proteins in the discovered pathways are being targeted to confirm our proteomic results and to test potential novel therapies that emulate GzmA. It will also be investigated if GzmA co-localizes in these cellular compartments. These studies will lead to a better understanding of GzmA biology during infection and to the development of novel host-directed therapeutics for control of Mtb infection.
Abstract
One fourth of the world population is infected with Mycobacterium tuberculosis (Mtb). Currently, BCG strains are the only vaccines available to protect against tuberculosis (TB). Our lab has ...identified γδ T cells that secrete Granzyme A (GzmA) with TB protective effects. GzmA when released by TB-specific γδ T cells has been shown to inhibit the intracellular replication of the pathogen. To elucidate the mechanism(s) involved in this inhibition, the lab has purified human GzmA. We have proven that Granzyme A’s intact active site is necessary to inhibit the intracellular replication of mycobacteria. The substitution of serine to alanine in the active site rendered the protein unable to control the mycobacterial infection as measured by mycobacterial growth inhibition assays, and the measurement of the pro-inflammatory marker TNF. Conversely, the effects of highly pure and active GzmA on mycobacterial replication inside human macrophages have been investigated using systems biology. A 2D-DIGE proteomic approach using adherent human monocytes has been performed to identify the pathways stimulated by GzmA involved in control of mycobacterial replication. Our analysis indicates that ER-stress responses (HSPA5, HSP90B1, P4HB, PDIA3) and purinergic channel receptor activation (ATP5B/C1/D/H/O) may be important for GzmA-mediated intracellular mycobacterial inhibition. Modulation of key proteins in these pathways are currently being targeted by pharmaceutical intervention and gene alteration to test the effects on GzmA-mediated inhibitory effects. Confirmation that these pathways are involved in pathogen clearance will lead to the development of novel host-directed therapies for control of Mtb infection.
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