Illuminating an Invisible Epidemic: A Systemic Review of the Clinical and Economic Benefits of Early Diagnosis and Treatment in Inflammatory Disease and Related Syndromes

Healthcare expenditures in the United States are growing at an alarming level with the Centers for Medicare and Medicaid Services (CMS) projecting that they will reach 5.7 trillion per year by 2026. Inflammatory diseases and related syndromes are growing in prevalence among Western societies. This growing population that affects close to 60 million people in the U.S. places a significant burden on the healthcare system. Characterized by relatively slow development, these diseases and syndromes prove challenging to diagnose, leading to delayed treatment against the backdrop of inevitable disability progression. Patients require healthcare attention but are initially hidden from clinician’s view by the seemingly generalized, non-specific symptoms. It is imperative to identify and manage these underlying conditions to slow disease progression and reduce the likelihood that costly comorbidities will develop. Enhanced diagnostic criteria coupled with additional technological innovation to identify inflammatory conditions earlier is necessary and in the best interest of all healthcare stakeholders. The current total cost to the U.S. healthcare system is at least 90B dollars annually. Through unique analysis of financial cost drivers, this review identifies opportunities to improve clinical outcomes and help control these disease-related costs by 20% or more

Survival, bacterial clearance and thrombocytopenia are improved in polymicrobial sepsis by targeting nuclear transport shuttles.

The rising tide of sepsis, a leading cause of death in the US and globally, is not adequately controlled by current antimicrobial therapies and supportive measures, thereby requiring new adjunctive treatments. Severe microvascular injury and multiple organ failure in sepsis are attributed to a "genomic storm" resulting from changes in microbial and host genomes encoding virulence factors and endogenous inflammatory mediators, respectively. This storm is mediated by stress-responsive transcription factors that are ferried to the nucleus by nuclear transport shuttles importins/karyopherins. We studied the impact of simultaneously targeting two of these shuttles, importin alpha 5 (Imp α5) and importin beta 1 (Imp β1), with a cell-penetrating Nuclear Transport Modifier (NTM) in a mouse model of polymicrobial sepsis. NTM reduced nuclear import of stress-responsive transcription factors nuclear factor kappa B, signal transducer and activator of transcription 1 alpha, and activator protein 1 in liver, which was also protected from sepsis-associated metabolic changes. Strikingly, NTM without antimicrobial therapy improved bacterial clearance in blood, spleen, and lungs, wherein a 700-fold reduction in bacterial burden was achieved while production of proinflammatory cytokines and chemokines in blood plasma was suppressed. Furthermore, NTM significantly improved thrombocytopenia, a prominent sign of microvascular injury in sepsis, inhibited neutrophil infiltration in the liver, decreased L-selectin, and normalized plasma levels of E-selectin and P-selectin, indicating reduced microvascular injury. Importantly, NTM combined with antimicrobial therapy extended the median time to death from 42 to 83 hours and increased survival from 30% to 55% (p = 0.022) as compared to antimicrobial therapy alone. This study documents the fundamental role of nuclear signaling mediated by Imp α5 and Imp β1 in the mechanism of polymicrobial sepsis and highlights the potential for targeting nuclear transport as an adjunctive therapy in sepsis management

The "genomic storm" induced by bacterial endotoxin is calmed by a nuclear transport modifier that attenuates localized and systemic inflammation.

Lipopolysaccharide (LPS) is a potent microbial virulence factor that can trigger production of proinflammatory mediators involved in the pathogenesis of localized and systemic inflammation. Importantly, the role of nuclear transport of stress responsive transcription factors in this LPS-generated "genomic storm" remains largely undefined. We developed a new nuclear transport modifier (NTM) peptide, cell-penetrating cSN50.1, which targets nuclear transport shuttles importin α5 and importin β1, to analyze its effect in LPS-induced localized (acute lung injury) and systemic (lethal endotoxic shock) murine inflammation models. We analyzed a human genome database to match 46 genes that encode cytokines, chemokines and their receptors with transcription factors whose nuclear transport is known to be modulated by NTM. We then tested the effect of cSN50.1 peptide on proinflammatory gene expression in murine bone marrow-derived macrophages stimulated with LPS. This NTM suppressed a proinflammatory transcriptome of 37 out of 84 genes analyzed, without altering expression of housekeeping genes or being cytotoxic. Consistent with gene expression analysis in primary macrophages, plasma levels of 23 out of 26 LPS-induced proinflammatory cytokines, chemokines, and growth factors were significantly attenuated in a murine model of LPS-induced systemic inflammation (lethal endotoxic shock) while the anti-inflammatory cytokine, interleukin 10, was enhanced. This anti-inflammatory reprogramming of the endotoxin-induced genomic response was accompanied by complete protection against lethal endotoxic shock with prophylactic NTM treatment, and 75% protection when NTM was first administered after LPS exposure. In a murine model of localized lung inflammation caused by direct airway exposure to LPS, expression of cytokines and chemokines in the bronchoalveolar space was suppressed with a concomitant reduction of neutrophil trafficking. Thus, calming the LPS-triggered "genomic storm" by modulating nuclear transport with cSN50.1 peptide attenuates the systemic inflammatory response associated with lethal shock as well as localized lung inflammation

NTM treatment enhances survival from lethal endotoxic shock.

<p>Survival curves for mice challenged i.p. with high-dose LPS (A and B) or low-dose LPS+D-Gal (C). In (A) and (C) mice were administered the first NTM (cSN50.1 peptide) treatment 30 min before LPS challenge (prophylactic protocol), while in (B), the first treatment was administered 15 min after LPS challenge (therapeutic protocol). Saline injections were administered to control mice challenged with LPS following the same treatment schedule in each protocol, as described in “Materials and Methods”. ***<i>p</i><0.0005 by log rank test.</p

Survival is increased by combining NTM treatment with antibiotic therapy.

<p>Mice were infected with CS and treated with vehicle or NTM (cSN50.1), both supplemented by antibiotic therapy with meropenem (<i>n</i> = 20 mice/group; Kaplan-Meier survival plot with <i>p</i> value calculated by log rank analysis).</p

Amino acid sequence of cSN50.1 peptide and its congeners SN50 and cSN50.

<p>Fragment-linked peptides comprising the Signal Sequence Hydrophobic Region of Fibroblast Growth Factor 4 (bolded) and the NLS region of NFκB1/p50 (italicized) were analyzed for their solubility in water. In cSN50 and cSN50.1, an intra-molecular disulfide bond is formed between the two cysteines, which cyclizes the NLS motif.</p><p>Amino acid sequence of cSN50.1 peptide and its congeners SN50 and cSN50.</p

LPS-induced cellular trafficking to lungs is reduced in NTM-treated mice.

<p>Cell counts in BAL fluid collected from unchallenged mice (naïve) and 6 h after direct airway exposure to LPS, with i.p. NTM peptide (cSN50.1) or diluent control (saline) treatment. The LPS-induced increase in total cells is comprised primarily of neutrophils. Neutrophil trafficking to BAL is significantly reduced by NTM treatment while monocytes/macrophages and lymphocytes are not affected. Data are presented as mean ± standard error, <i>n</i> = 4 naïve and 5–7 NTM- or saline-treated animals/group from two independent experiments. *<i>p</i><0.05, **<i>p</i><0.005 by Mann-Whitney <i>U</i> test comparing LPS-challenged groups.</p

NTM treatment reduces plasma levels of multiple cytokines, chemokines and growth factors induced by LPS.

<p>(A) Wild type C57BL/6 mice were challenged i.p. with a lethal dose of LPS (800 µg) and treated with i.p. injections of NTM (cSN50.1 peptide) or diluent (saline) following a prophylactic protocol as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110183#pone-0110183-g004" target="_blank">Figure 4A</a>. Blood was collected at baseline and 2 or 6 h after LPS challenge and a multiplex assay was used to measure 32 analytes in plasma. Twenty-four analytes were significantly altered by NTM treatment, as determined by repeated measures two-way analysis of variance with Sidak’s post-test. Twenty-three were reduced, while anti-inflammatory IL-10 was increased by NTM treatment. Results are shown as the % inhibition or increase by NTM compared to saline control set to 100% at the time point demonstrating maximal expression for that analyte. <i>n</i> = 10 animals/group. (B) Comparison of prophylactic and therapeutic NTM treatment protocols on selected plasma cytokine and chemokine levels in the high-dose LPS model of endotoxic shock. Data are presented as mean ± standard error, <i>n</i> = 5 −10 animals/group.</p

Bacterial clearance is improved in infected mice treated with NTM.

<p>CFU determined by serial dilution of whole blood and organ homogenates collected 12 h after infection from sham- or CS-infected mice treated with NTM (cSN50.1) or vehicle. Bars represent median values from 4–5 mice/group (<i>p</i> values determined by Mann-Whitney test).</p

LPS-induced expression of chemokines, cytokines, and growth factors in the lung is suppressed by NTM.

<p>Fourteen cytokines, chemokines and growth factors elevated in BAL after direct airway exposure to LPS are significantly suppressed by NTM (cSN50.1 peptide) treatment. Data are presented as mean ± standard error, <i>n</i> = 4 naïve and 8–9 NTM- or saline-treated animals/group from 3 independent experiments. *<i>p</i><0.05, **<i>p</i><0.005, and ***<i>p</i><0.0005 by Mann-Whitney <i>U</i> test comparing LPS-challenged groups.</p