Hematological Trends in Severe Burn Patients: A Comprehensive Study for Prognosis and Clinical Insights.

Severe burn injuries pose diagnostic challenges, contributing to increased fatality rates with delayed diagnoses. This study aims to identify early risk factors and understand their impact on clinical outcomes by examining hematological dynamics in severe burn cases. The focus includes age-related patterns, Total Body Surface Area (TBSA) affected by burns, hospital stay duration, and changes in hematological markers during burn injuries. An analytical cross-sectional study at the Burn Care Centre involved 135 participants hospitalized between January 2018 and December 2021. Demographic data and hematological markers were recorded, with statistical analysis using IBM SPSS 25.0. Non-survivors exhibited a greater mean TBSA, shorter hospital stay, and an enhanced early immune response indicated by WBC count on the first day. Hematological markers, including HGB, RCC, and PLT, showed dynamic patterns over the study period. Marginal variations in platelet counts and intriguing patterns in RCC suggested potential consequences like disseminated intravascular coagulation. The study provides crucial insights into hematological responses to severe burn injuries. Early identification of risk factors, particularly age-related patterns and immune responses, informs clinicians about predicting outcomes and guiding therapeutic interventions. Despite limitations, this work underscores the need for further multi-center research to comprehensively understand the complex relationships between burn injuries, hematological responses, and clinical outcomes.

Preparing for the future of public health: ecological determinants of health and the call for an eco-social approach to public health education.

As a collective organized to address the education implications of calls for public health engagement on the ecological determinants of health, we, the Ecological Determinants Group on Education (cpha.ca/EDGE), urge the health community to properly understand and address the importance of the ecological determinants of the public's health, consistent with long-standing calls from many quarters-including Indigenous communities-and as part of an eco-social approach to public health education, research and practice. Educational approaches will determine how well we will be equipped to understand and respond to the rapid changes occurring for the living systems on which all life-including human life-depends. We revisit findings from the Canadian Public Health Association's discussion paper on 'Global Change and Public Health: Addressing the Ecological Determinants of Health', and argue that an intentionally eco-social approach to education is needed to better support the health sector's role in protecting and promoting health, preventing disease and injury, and reducing health inequities. We call for a proactive approach, ensuring that the ecological determinants of health become integral to public health education, practice, policy, and research, as a key part of wider societal shifts required to foster a healthy, just, and ecologically sustainable future.

Tumor Necrosis Factor-α Increases Claudin-1, 4, and 7 Expression in Tubular Cells: Role in Permeability Changes.

Tumor necrosis factor-α (TNFα), is a pathogenic cytokine in kidney disease that alters expression of claudins in tubular cells. Previously we showed that in LLC-PK1 cells TNFα caused a biphasic change in transepithelial resistance (TER) consisting of an early drop and recovery, followed by a late increase. However, the underlying mechanisms and the role of specific claudins in the TER effect remained incompletely understood. Here we sought to define how TNFα affects claudins 1, 4, and 7 in tubular cells and to correlate their changes with the TER effect. We show that TNFα elevates total and surface levels of Cldn-1, 4, and 7, and increases their mRNA expression through the ERK and JNK pathways. Further, JNK is also important for TNFα-induced changes in claudin-2 expression. Continuous monitoring of TER using Electric cell-substrate impedance sensing (ECIS) reveals that the two phases of the TNFα effect are differently regulated. Specifically, inhibition of the ERK or JNK pathways prevent the late TER increase, but not the early TER effect. Silencing experiments also show that Cldn-1 is necessary for the early TNFα-induced TER change, while all three claudins appear to contribute to the late TER increase. In summary, we define a central role for ERK and JNK in TNFα-induced altered claudin expression and barrier tightening. Together, our current and previous works show that the TNFα-induced early TER effect requires claudin-1, while claudin-2 decrease is a significant mediator of the late TER increase, and elevation in claudin-1, 4, and 7 contribute to a smaller extent. J. Cell. Physiol. 232: 2210-2220, 2017. © 2016 Wiley Periodicals, Inc.

Tumor necrosis factor-α induces a biphasic change in claudin-2 expression in tubular epithelial cells: role in barrier functions.

The inflammatory cytokine tumor necrosis factor-α (TNF-α) is a pathogenic factor in acute and chronic kidney disease. TNF-α is known to alter expression of epithelial tight junction (TJ) proteins; however, the underlying mechanisms and the impact of this effect on epithelial functions remain poorly defined. Here we describe a novel biphasic effect of TNF-α on TJ protein expression. In LLC-PK1 tubular cells, short-term (1-6 h) TNF-α treatment selectively elevated the expression of the channel-forming TJ protein claudin-2. In contrast, prolonged (>8 h) TNF-α treatment caused a marked downregulation in claudin-2 and an increase in claudin-1, -4, and -7. The early increase and the late decrease in claudin-2 expression involved distinct mechanisms. TNF-α slowed claudin-2 degradation through ERK, causing the early increase. This increase was also mediated by the EGF receptor and RhoA and Rho kinase. In contrast, prolonged TNF-α treatment reduced claudin-2 mRNA levels and promoter activity independent from these signaling pathways. Electric Cell-substrate Impedance Sensing measurements revealed that TNF-α also exerted a biphasic effect on transepithelial resistance (TER) with an initial decrease and a late increase. Thus there was a good temporal correlation between TNF-α-induced claudin-2 protein and TER changes. Indeed, silencing experiments showed that the late TER increase was at least in part caused by reduced claudin-2 expression. Surprisingly, however, claudin-2 silencing did not prevent the early TER drop. Taken together, the TNF-α-induced changes in claudin-2 levels might contribute to TER changes and could also play a role in newly described functions of claudin-2 such as proliferation regulation.

Central role of the exchange factor GEF-H1 in TNF-α-induced sequential activation of Rac, ADAM17/TACE, and RhoA in tubular epithelial cells.

Transactivation of the epidermal growth factor receptor (EGFR) by tumor necrosis factor-α (TNF-α) is a key step in mediating RhoA activation and cytoskeleton and junction remodeling in the tubular epithelium. In this study we explore the mechanisms underlying TNF-α-induced EGFR activation. We show that TNF-α stimulates the TNF-α convertase enzyme (TACE/a disintegrin and metalloproteinase-17), leading to activation of the EGFR/ERK pathway. TACE activation requires the mitogen-activated protein kinase p38, which is activated through the small GTPase Rac. TNF-α stimulates both Rac and RhoA through the guanine nucleotide exchange factor (GEF)-H1 but by different mechanisms. EGFR- and ERK-dependent phosphorylation at the T678 site of GEF-H1 is a prerequisite for RhoA activation only, whereas both Rac and RhoA activation require GEF-H1 phosphorylation on S885. Of interest, GEF-H1-mediated Rac activation is upstream from the TACE/EGFR/ERK pathway and regulates T678 phosphorylation. We also show that TNF-α enhances epithelial wound healing through TACE, ERK, and GEF-H1. Taken together, our findings can explain the mechanisms leading to hierarchical activation of Rac and RhoA by TNF-α through a single GEF. This mechanism could coordinate GEF functions and fine-tune Rac and RhoA activation in epithelial cells, thereby promoting complex functions such as sheet migration.

Hyperosmotic stress regulates the distribution and stability of myocardin-related transcription factor, a key modulator of the cytoskeleton.

Hyperosmotic stress initiates several adaptive responses, including the remodeling of the cytoskeleton. Besides maintaining structural integrity, the cytoskeleton has emerged as an important regulator of gene transcription. Myocardin-related transcription factor (MRTF), an actin-regulated coactivator of serum response factor, is a major link between the actin skeleton and transcriptional control. We therefore investigated whether MRTF is regulated by hyperosmotic stress. Here we show that hypertonicity induces robust, rapid, and transient translocation of MRTF from the cytosol to the nucleus in kidney tubular cells. We found that the hyperosmolarity-triggered MRTF translocation is mediated by the RhoA/Rho kinase (ROK) pathway. Moreover, the Rho guanine nucleotide exchange factor GEF-H1 is activated by hyperosmotic stress, and it is a key contributor to the ensuing RhoA activation and MRTF translocation, since siRNA-mediated GEF-H1 downregulation suppresses these responses. While the osmotically induced RhoA activation promotes nuclear MRTF accumulation, the concomitant activation of p38 MAP kinase mitigates this effect. Moderate hyperosmotic stress (600 mosM) drives MRTF-dependent transcription through the cis-element CArG box. Silencing or pharmacological inhibition of MRTF prevents the osmotic stimulation of CArG-dependent transcription and renders the cells susceptible to osmotic shock-induced structural damage. Interestingly, strong hyperosmolarity promotes proteasomal degradation of MRTF, concomitant with apoptosis. Thus, MRTF is an osmosensitive and osmoprotective transcription factor, whose intracellular distribution is regulated by the GEF-H1/RhoA/ROK and p38 pathways. However, strong osmotic stress destabilizes MRTF, concomitant with apoptosis, implying that hyperosmotically induced cell death takes precedence over epithelial-myofibroblast transition, a potential consequence of MRTF-mediated phenotypic reprogramming.

Affinity precipitation of active Rho-GEFs using a GST-tagged mutant Rho protein (GST-RhoA(G17A)) from epithelial cell lysates.

Proteins of the Rho family of small GTPases are central regulators of the cytoskeleton, and control a large variety of cellular processes, including cell migration, gene expression, cell cycle progression and cell adhesion. Rho proteins are molecular switches that are active in GTP-bound and inactive in GDP-bound state. Their activation is mediated by a family of Guanine-nucleotide Exchange Factor (GEF) proteins. Rho-GEFs constitute a large family, with overlapping specificities. Although a lot of progress has been made in identifying the GEFs activated by specific signals, there are still many questions remaining regarding the pathway-specific regulation of these proteins. The number of Rho-GEFs exceeds 70, and each cell expresses more than one GEF protein. In addition, many of these proteins activate not only Rho, but other members of the family, contributing further to the complexity of the regulatory networks. Importantly, exploring how GEFs are regulated requires a method to follow the active pool of individual GEFs in cells activated by different stimuli. Here we provide a step-by-step protocol for a method used to assess and quantify the available active Rho-specific GEFs using an affinity precipitation assay. This assay was developed a few years ago in the Burridge lab and we have used it in kidney tubular cell lines. The assay takes advantage of a "nucleotide free" mutant RhoA, with a high affinity for active GEFs. The mutation (G17A) renders the protein unable to bind GDP or GTP and this state mimics the intermediate state that is bound to the GEF. A GST-tagged version of this mutant protein is expressed and purified from E. coli, bound to glutathione sepharose beads and used to precipitate active GEFs from lysates of untreated and stimulated cells. As most GEFs are activated via posttranslational modifications or release from inhibitory bindings, their active state is preserved in cell lysates, and they can be detected by this assay. Captured proteins can be probed for known GEFs by detection with specific antibodies using Western blotting, or analyzed by Mass Spectrometry to identify unknown GEFs activated by certain stimuli.

RhoA and Rho kinase mediate cyclosporine A and sirolimus-induced barrier tightening in renal proximal tubular cells.

The regulation and maintenance of the paracellular transport in renal tubular epithelia is vital for kidney functions. Combination of the immunosuppressant drugs cyclosporine A (CsA) and sirolimus (SRL) exerts powerful immunosuppression, but also causes nephrotoxicity. We have previously shown that CsA and SRL elevate transepithelial resistance (TER) in kidney tubular cells partly through MEK/ERK1/2. In this work we examined the hypothesis that the RhoA pathway may also be mediating effects of CsA and SRL. We show that CsA and the CsA/SRL combination activated RhoA, induced cofilin phosphorylation and promoted stress fiber generation. The Rho kinase (ROK) inhibitor, Y27632, prevented CsA and CsA/SRL-induced cofilin phosphorylation and actin remodelling, reduced the TER increase and prevented the rise in claudin-7 levels caused by the drugs. Expression of the exchange factor GEF-H1/lfc was elevated in cells treated with CsA and CsA/SRL. GEF-H1 silencing inhibited RhoA activation by ≈50%, and potently reduced cofilin phosphorylation and stress fiber formation induced by CsA and CsA/SRL. However, GEF-H1 downregulation did not prevent the TER change. Thus the Rho/Rho kinase pathway was involved in mediating CsA and CsA/SRL-induced cytoskeleton rearrangement and TER changes via claudin-7 expression. Our data however point to differential regulation of Rho activation involved in central cytoskeleton remodelling, that is GEF-H1-dependent and junctional permeability that does not require GEF-H1.

Pre-B cell colony-enhancing factor (PBEF/Nampt/visfatin) primes neutrophils for augmented respiratory burst activity through partial assembly of the NADPH oxidase.

Pre-B cell colony-enhancing factor ([PBEF] also known as Nampt/visfatin) is a pleiotropic 52-kDa cytokine-like molecule whose activity has been implicated in multiple inflammatory disease states. PBEF promotes polymorphonuclear neutrophil (PMN) proinflammatory function by inhibiting constitutive PMN apoptosis. We investigated whether PBEF activates or primes for PMN respiratory burst. We found that although PBEF did not activate respiratory burst on its own, it primed for increased reactive oxygen species generation through the NADPH oxidase. PBEF promoted membrane translocation of cytosolic NADPH oxidase subunits p40 and p47, but not p67, induced p40 phosphorylation on Thr(154), and activated the small GTPase Rac. Priming, translocation, and phosphorylation were dependent on activation of p38 and ERK MAPKs, but not of PI3K. Priming by PBEF occurred independent of its NAD-generating capacity because neither nicotinamide mononucleotide or NAD could recapitulate the effects, and a specific inhibitor of PBEF, APO-866, could not inhibit priming. Taken together, these results demonstrate that PBEF can prime for PMN respiratory burst activity by promoting p40 and p47 translocation to the membrane, and this occurs in a MAPK-dependent fashion.

The epidermal growth factor receptor mediates tumor necrosis factor-alpha-induced activation of the ERK/GEF-H1/RhoA pathway in tubular epithelium.

Tumor necrosis factor (TNF)-α induces cytoskeleton and intercellular junction remodeling in tubular epithelial cells; the underlying mechanisms, however, are incompletely explored. We have previously shown that ERK-mediated stimulation of the RhoA GDP/GTP exchange factor GEF-H1/Lfc is critical for TNF-α-induced RhoA stimulation. Here we investigated the upstream mechanisms of ERK/GEF-H1 activation. Surprisingly, TNF-α-induced ERK and RhoA stimulation in tubular cells were prevented by epidermal growth factor receptor (EGFR) inhibition or silencing. TNF-α also enhanced phosphorylation of the EGFR. EGF treatment mimicked the effects of TNF-α, as it elicited potent, ERK-dependent GEF-H1 and RhoA activation. Moreover, EGF-induced RhoA activation was prevented by GEF-H1 silencing, indicating that GEF-H1 is a key downstream effector of the EGFR. The TNF-α-elicited EGFR, ERK, and RhoA stimulation were mediated by the TNF-α convertase enzyme (TACE) that can release EGFR ligands. Further, EGFR transactivation also required the tyrosine kinase Src, as Src inhibition prevented TNF-α-induced activation of the EGFR/ERK/GEF-H1/RhoA pathway. Importantly, a bromodeoxyuridine (BrdU) incorporation assay and electric cell substrate impedance-sensing (ECIS) measurements revealed that TNF-α stimulated cell growth in an EGFR-dependent manner. In contrast, TNF-α-induced NFκB activation was not prevented by EGFR or Src inhibition, suggesting that TNF-α exerts both EGFR-dependent and -independent effects. In summary, in the present study we show that the TNF-α-induced activation of the ERK/GEF-H1/RhoA pathway in tubular cells is mediated through Src- and TACE-dependent EGFR activation. Such a mechanism could couple inflammatory and proliferative stimuli and, thus, may play a key role in the regulation of wound healing and fibrogenesis.

Extracellular signal-regulated kinase and GEF-H1 mediate depolarization-induced Rho activation and paracellular permeability increase.

Plasma membrane depolarization activates the Rho/Rho kinase (ROK) pathway and thereby enhances myosin light chain (MLC) phosphorylation, which in turn is thought to be a key regulator of paracellular permeability. However, the upstream mechanisms that couple depolarization to Rho activation and permeability changes are unknown. Here we show that three different depolarizing stimuli (high extracellular K(+) concentration, the lipophilic cation tetraphenylphosphonium, or l-alanine, which is taken up by electrogenic Na(+) cotransport) all provoke robust phosphorylation of ERK in LLC-PK1 and Madin-Darby canine kidney (MDCK) cells. Importantly, inhibition of ERK prevented the depolarization-induced activation of Rho. Searching for the underlying mechanism, we have identified the GTP/GDP exchange factor GEF-H1 as the ERK-regulated critical exchange factor responsible for the depolarization-induced Rho activation. This conclusion is based on our findings that 1) depolarization activated GEF-H1 but not p115RhoGEF, 2) short interfering RNA-mediated GEF-H1 silencing eliminated the activation of the Rho pathway, and 3) ERK inhibition prevented the activation of GEF-H1. Moreover, we found that the Na(+)-K(+) pump inhibitor ouabain also caused ERK, GEF-H1, and Rho activation, partially due to its depolarizing effect. Regarding the functional consequences of this newly identified pathway, we found that depolarization increased paracellular permeability in LLC-PK1 and MDCK cells and that this effect was mitigated by inhibiting myosin using blebbistatin or a dominant negative (phosphorylation incompetent) MLC. Taken together, we propose that the ERK/GEF-H1/Rho/ROK/pMLC pathway could be a central mechanism whereby electrogenic transmembrane transport processes control myosin phosphorylation and regulate paracellular transport in the tubular epithelium.

GEF-H1 mediates tumor necrosis factor-alpha-induced Rho activation and myosin phosphorylation: role in the regulation of tubular paracellular permeability.

Tumor necrosis factor-alpha (TNF-alpha), an inflammatory cytokine, has been shown to activate the small GTPase Rho, but the underlying signaling mechanisms remained undefined. This general problem is particularly important in the kidney, because TNF-alpha, a major mediator of kidney injury, is known to increase paracellular permeability in tubular epithelia. Here we aimed to determine the effect of TNF-alpha on the Rho pathway in tubular cells (LLC-PK(1) and Madin-Darby canine kidney), define the upstream signaling, and investigate the role of the Rho pathway in the TNF-alpha-induced alterations of paracellular permeability. We show that TNF-alpha induced a rapid and sustained RhoA activation that led to stress fiber formation and Rho kinase-dependent myosin light chain (MLC) phosphorylation. To identify new regulators connecting the TNF receptor to Rho signaling, we applied an affinity precipitation assay with a Rho mutant (RhoG17A), which captures activated GDP-GTP exchange factors (GEFs). Mass spectrometry analysis of the RhoG17A-precipitated proteins identified GEF-H1 as a TNF-alpha-activated Rho GEF. Consistent with a central role of GEF-H1, its down-regulation by small interfering RNA prevented the activation of the Rho pathway. Moreover GEF-H1 and Rho activation are downstream of ERK signaling as the MEK1/2 inhibitor PD98059 mitigated TNF-alpha-induced activation of these proteins. Importantly TNF-alpha enhanced the ERK pathway-dependent phosphorylation of Thr-678 of GEF-H1 that was key for activation. Finally the TNF-alpha-induced paracellular permeability increase was absent in LLC-PK(1) cells stably expressing a non-phosphorylatable, dominant negative MLC. In summary, we have identified the ERK/GEF-H1/Rho/Rho kinase/phospho-MLC pathway as the mechanism mediating TNF-alpha-induced elevation of tubular epithelial permeability, which in turn might contribute to kidney injury.