A pharmacist-led penicillin allergy de-labelling project within a preoperative assessment clinic: the low-hanging fruit is within reach.

BACKGROUND: Having a false penicillin-allergy label is linked to longer hospital stays and to an increased risk of Clostridioides difficile and meticillin-resistant Staphylococcus aureus infection. AIM: To assess a penicillin-allergy de-labelling tool designed for use by the non-allergist. METHODS: Patients attending the surgical preoperative assessment clinic (POAC) at a large UK teaching hospital, who reported a penicillin allergy, were directly de-labelled by nursing or pharmacy staff, where appropriate. A penicillin-allergy de-labelling tool designed for use by the non-allergist was adapted and applied; nursing staff were provided with supporting information and education to enable removal of spurious labels. Antimicrobial pharmacists (AMPs) provided follow-up, cross-checked prophylactic antibiotics administered, interrogated clinical notes, and telephoned patients following their surgery, for details of any adverse reactions suffered. FINDINGS: A total of 163 patients reporting a penicillin allergy were identified for intervention. Twenty-nine (17.8%) patients reported a penicillin-allergy history appropriate for direct de-labelling, of whom eight (27.6%) declined to consent. The remaining 21 patients (12.8%) were directly de-labelled, with 12 (7.4%) patients consenting during their POAC appointment; the remaining nine (5.5%) patients were consented and de-labelled after their surgery by an AMP. CONCLUSION: The POAC was identified as an appropriate location and time-point in the patient pathway to enable the direct removal of spurious penicillin-allergy labels prior to surgery. Results suggest that this could be undertaken by nursing staff, although support from AMPs enabled a greater number of patients to be de-labelled.

The spectrum of hemophagocytic lymphohistiocytosis: a retrospective study comparing adult macrophage activation syndrome to malignancy-associated hemophagocytic lymphohistiocytosis.

Secondary hemophagocytic lymphohistiocytosis (sHLH) is a life-threatening inflammatory syndrome that can be triggered by autoimmune diseases, malignancy, or infection. In rheumatologic patients, sHLH is referred to as macrophage activation syndrome (MAS). Differentiating between triggers is important for prompt treatment and prognosis. Data comparing subsets of sHLH are limited due to the rarity of this disease. We aim to explore differences in clinical features that may differentiate MAS from malignancy-associated HLH (mHLH) patients. We conducted a single-center retrospective study assessing clinical characteristics, laboratory parameters, treatment regimens and outcomes in 34 patients with sHLH over a 16 year period. We compared patients with MAS to those with mHLH. Hepatomegaly was not present in the MAS group but was present in the mHLH group (0 vs. 25%, p = 0.024). MAS patients had on average nearly double the concentration of platelets at 50.0 (IQR: 31.0-78.0 Kµ/L) vs. 29.0 Kµ/L (IQR: 14.0-37.5 Kµ/L), p = 0.003. Soluble IL-2R concentrations were four times lower in the MAS group with a median soluble IL-2R concentration of 6814.5 kU/L (IQR: 2101-2610 kU/L) vs. 27972.0 kU/L (IQR: 12,820-151,650 kU/L), p = 0.010. The MAS group fared better overall than the mHLH group but was not statistically significant (mortality 22 vs. 44%, p = 0.18). MAS and mHLH patients exhibited different laboratory parameters and clinical features, most notably differences in platelet counts, soluble IL-2R concentration and hepatomegaly, which may help differentiate these conditions early in their course.

Autonomic Regulation of Nociceptive and Immunologic Changes in a Mouse Model of Complex Regional Pain Syndrome.

Chronic pain frequently develops after limb injuries, and its pathogenesis is poorly understood. We explored the hypothesis that the autonomic nervous system regulates adaptive immune system activation and nociceptive sensitization in a mouse model of chronic post-traumatic pain with features of complex regional pain syndrome (CRPS). In studies sympathetic signaling was reduced using 6-hydroxydopamine (6-OHDA) or lofexidine, while parasympathetic signaling was augmented by nicotine administration. Hindpaw allodynia, unweighting, skin temperature, and edema were measured at 3 and 7 weeks after fracture. Hypertrophy of regional lymph nodes and IgM deposition in the skin of injured limbs were followed as indices of adaptive immune system activation. Passive transfer of serum from fracture mice to recipient B cell deficient (muMT) mice was used to assess the formation of pain-related autoantibodies. We observed that 6-OHDA or lofexidine reduced fracture-induced hindpaw nociceptive sensitization and unweighting. Nicotine had similar effects. These treatments also prevented IgM deposition, hypertrophy of popliteal lymph nodes, and the development of pronociceptive serum transfer effects. We conclude that inhibiting sympathetic or augmenting parasympathetic signaling inhibits pro-nociceptive immunological changes accompanying limb fracture. These translational results support the use of similar approaches in trials potentially alleviating persistent post-traumatic pain and, possibly, CRPS. PERSPECTIVE: Selective treatments aimed at autonomic nervous system modulation reduce fracture-related nociceptive and functional sequelae. The same treatment strategies limit pain-supporting immune system activation and the production of pro-nociceptive antibodies. Thus, the therapeutic regulation of autonomic activity after limb injury may reduce the incidence of chronic pain.

Unmasking latent thioesters under hydrophobic-compatible conditions.

Hydrophobic latent C-terminal thioesters were converted into thioesters, and were also coupled with cysteine in one-pot reactions, using conditions generally compatible with hydrophobic materials. The reaction conditions (ethanethiol and triethylamine in a mixture of DMF and THF) are compatible with acid-labile protecting groups (Boc/t-Bu) that are standard in Fmoc peptide synthesis.

Computed tomography perfusion abnormalities after carotid endarterectomy help in the diagnosis of reversible cerebral vasoconstriction syndrome.

Acute neurologic deficits in the postoperative period after carotid endarterectomy (CEA) can prompt extensive diagnostic evaluation. Reversible cerebral vasoconstriction syndrome (RCVS) is an underrecognized cause of acute neurologic deficit after CEA. We present the case of RCVS in an 84-year-old male patient who had experienced left limb weakness after CEA, prompting multiple code stroke activations. The present case is novel because the obtained computed tomography perfusion imaging studies demonstrated abnormalities that have not been previously described in patients with RCVS. These findings, combined with the cerebral angiography findings, led to the rapid diagnosis and delivery of intra-arterial vasodilator therapy. He experienced subsequent resolution of his symptoms and radiologic abnormalities.

Dimethyl Fumarate Reduces Oxidative Stress and Pronociceptive Immune Responses in a Murine Model of Complex Regional Pain Syndrome.

BACKGROUND: Complex regional pain syndrome (CRPS) is a highly disabling cause of pain often precipitated by surgery or trauma to a limb. Both innate and adaptive immunological changes contribute to this syndrome. Dimethyl fumarate (DMF) works through the nuclear factor erythroid 2-related factor 2 (Nrf2) transcription factor and other targets to activate antioxidant systems and to suppress immune system activation. We hypothesized that DMF would reduce nociceptive, functional, and immunological changes measured in a model of CRPS. METHODS: Male C57BL/6 mice were used in the well-characterized tibial fracture model of CRPS. Some groups of mice received DMF 25 mg/kg/d orally, per os for 3 weeks after fracture versus vehicle alone. Homozygous Nrf2 null mutant mice were used as test subjects to address the need for this transcription factor for DMF activity. Allodynia was assessed using von Frey filaments and hindlimb weight-bearing data were collected. The markers of oxidative stress malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE) were quantified in the skin of the fractured mice using immunoassays along with the innate immune system cytokines IL-1ß and IL-6. The accumulation of IgM in the fractured limbs and lymph node hypertrophy were used as indexes of adaptive immune system activation, and the passive transfer of serum from wildtype fractured mice to B cell-deficient fractured muMT mice (mice lacking B cells and immunoglobulin) helped to assess the pronociceptive activity of humoral factors. RESULTS: We observed that oral DMF administration strongly prevented nociceptive sensitization and reduced uneven hindlimb weight bearing after fracture. DMF was also very effective in reducing the accumulation of markers of oxidative stress, activation of innate immune mediator production, lymph node hypertrophy, and the accumulation of IgM in fractured limbs. The sera of fractured vehicle-treated but not DMF-treated mice conferred pronociceptive activity to recipient mice. Unexpectedly, the effects of DMF were largely unchanged in the Nrf2 null mutant mice. CONCLUSIONS: Oxidative stress and immune system activation are robust after hindlimb fracture in mice. DMF strongly reduces activation of those systems, and the Nrf2 transcription factor is not required. DMF or drugs working through similar mechanisms might provide effective therapy for CRPS or other conditions where oxidative stress causes immune system activation.

The package as a weapon of influence: Changes to cigarette packaging design as a function of regulatory changes in Canada.

INTRODUCTION: Given existing regulations that ban the tobacco industry from engaging in traditional forms of advertising and require warning labels on cigarette packaging, we suggest that one response on the part of tobacco manufacturers has been to make alterations to design elements of cigarette packages themselves. The current research seeks to examine how cigarette manufacturers have altered elements of cigarette packaging in response to regulatory changes by the Government of Canada in 2011, which increased health warning sizes on cigarette packages from 50% of the principal display surface to 75%. METHODS: Cigarette packages (n=1689) that had been on the market in Canada in the period 2001-2017 were examined and coded for package design elements including package innovation (size and package style), color (hue and saturation), and branding elements (use of iconography and variant names). Characteristics of pre-regulation packaging were then systematically compared to characteristics of post-regulation packaging. RESULTS: Many of these packaging design elements, including package size and package style, primary and secondary hue, color saturation, use of variant label names, and use of iconography have systematically varied in response to regulatory changes in Canada. For example, we observed increases in the use of flip-top (vs slide and shell) packaging, the use of yellow, black and white as the focal color, incidence of color-themed variant names, and the use of female and crest-related logos. CONCLUSIONS: The evidence suggests that many packaging design elements have varied systematically along with regulatory changes in Canada.

Complex regional pain syndrome patient immunoglobulin M has pronociceptive effects in the skin and spinal cord of tibia fracture mice.

It has been proposed that complex regional pain syndrome (CRPS) is a post-traumatic autoimmune disease. Previously, we observed that B cells are required for the full expression of CRPS-like changes in a mouse tibia fracture model and that serum immunoglobulin M (IgM) antibodies from fracture mice have pronociceptive effects in muMT fracture mice lacking B cells. The current study evaluated the pronociceptive effects of injecting CRPS patient serum or antibodies into muMT fracture mice by measuring hind paw allodynia and unweighting changes. Complex regional pain syndrome serum binding was measured against autoantigens previously identified in the fracture mouse model. Both CRPS patient serum or IgM antibodies had pronociceptive effects in the fracture limb when injected systemically in muMT fracture mice, but normal subject serum and CRPS patient IgG antibodies had no effect. Furthermore, CRPS serum IgM antibodies had pronociceptive effects when injected into the fracture limb hind paw skin or intrathecally in the muMT fracture mice. Early (1-12 months after injury) CRPS patient (n = 20) sera were always pronociceptive after systemic injection, and chronic (>12 months after injury) CRPS sera were rarely pronociceptive (2/20 patients), while sera from normal subjects (n = 20) and from patients with uncomplicated recoveries from orthopedic surgery and/or fracture (n = 15) were never pronociceptive. Increased CRPS serum IgM binding was observed for keratin 16, histone 3.2, gamma actin, and alpha enolase autoantigens. We postulate that CRPS patient IgM antibodies bind to neoantigens in the fracture mouse skin and spinal cord to initiate a regionally restricted pronociceptive complement response potentially contributing to the CRPS disease process.

Validation and iteration of CT perfusion defined malignant profile thresholds for acute ischemic stroke.

BACKGROUND: Malignant profile computed tomography perfusion (CTP) lesions are associated with poor outcomes after administration of intravenous tissue-plasminogen activator (IV-tPA) for ischemic stroke. AIMS: To determine whether published CTP-based lesion thresholds predictive of poor outcomes in a predominantly 8 cm of CTP anatomic coverage cohort would predict poor outcomes in an independent 4 cm of CTP anatomic coverage cohort and to generate optimized 4 cm CTP thresholds. METHODS: Ischemic stroke patients with baseline CTP imaging with 4 cm of anatomic coverage before receiving IV-tPA at a single institution were retrospectively studied. Perfusion lesion time to maximum of tissue residue function (Tmax) and cerebral blood flow (CBF) volumes were determined using RAPID automated software. Fisher's exact tests assessed associations between lesion thresholds and outcomes. Receiver operating characteristic (ROC) curves generated optimized thresholds for 4 cm of CTP coverage. RESULTS: Sixty-three patients were included. Poor outcomes were associated with published thresholds of Tmax >6 s > 103 mL, Tmax > 8 s > 86 mL, and Tmax > 10 s > 78 mL but not CBF core >53 mL. Thresholds optimized for 4 cm of CTP coverage and associated with poor outcomes were Tmax > 6 s > 100 mL, Tmax > 8 s > 65 mL, Tmax >10 s > 46 mL, and CBF core >39 mL. CONCLUSIONS: We validated the ability of published CTP Tmax lesion volume thresholds to predict poor outcomes despite IV-tPA in an independent cohort using only 4 cm of CTP anatomical coverage. A CBF > 39 mL threshold, rather than the predominantly 8 cm CTP coverage derived CBF threshold of >53 mL, was associated with poor outcomes in this 4 cm CTP coverage cohort.

Enhanced angiogenic function in response to fibroblasts from psoriatic arthritis synovium compared to rheumatoid arthritis.

INTRODUCTION: Angiogenesis is an early event in the pathogenesis of both psoriatic arthritis (PsA) and rheumatoid arthritis (RA); however, there are striking differences in blood vessel morphology and activation between the two arthropathies. The aim of this study was to assess if the PsA and RA joint microenvironments differentially regulate endothelial cell function. METHODS: PsA and RA primary synovial fibroblasts (SFC) were isolated from synovial biopsies, grown to confluence, and supernatants harvested and termed 'conditioned media' (CM). Human umbilical vein endothelial cells (HUVEC) were cultured with PsA SFC or RA SFC-CM (20%). HUVEC tube formation, migration, and PBMC adhesion were assessed by matrigel tube formation, wound repair, and PBMC adhesion assays. HUVEC cell surface expression of ICAM, VCAM, and E-Selectin was assessed by flow cytometry. Transcriptome analysis of genes promoting angiogenesis was performed by real-time PCR. Finally, a MSD multiplex angiogenic assay was performed on PsA SFC and RA SFC supernatants. RESULTS: Macroscopic synovitis and vascularity were similar in PsA and RA patients; however, significant differences in vascular morphological pattern were recorded with tortuous, elongated vessels observed in PsA compared to straight regular branching vessels observed in RA. Transcriptome analysis showed strong upregulation of the pro-angiogenic signature in HUVEC primed with PsA SFC-CM compared to RA SFC-CM and basal control. In parallel, paired PsA SFC-CM significantly induced HUVEC tube formation compared to that of RA SFC-CM. Furthermore, PsA SFC-CM induced HUVEC migration was paralleled by a significant induction in VEGFA, PFKFB3, ICAM-1, and MMP3 mRNA expression. A significant increase in PBMC adhesion and cell surface expression of VCAM-1, ICAM-1, and E-Selectin expression was also demonstrated in PsA SFC-CM-primed HUVEC compared to RA SFC-CM. Finally, VEGF, TSLP, Flt-1, and Tie-2 expression was elevated in PsA SFC-CM compared to RA SFC-CM, with no significant difference in other pro-angiogenic mediators including MIP-3, bFGF, PIGF, and MCP-1. CONCLUSION: PsA SFC and RA SFC secreted factors differentially regulate endothelial cell function, with soluble mediators in the PsA joint microenvironment inducing a more pro-angiogenic phenotype compared to the RA.

Morphine Exacerbates Postfracture Nociceptive Sensitization, Functional Impairment, and Microglial Activation in Mice.

BACKGROUND: Emerging evidence suggests that opioid use immediately after surgery and trauma may worsen outcomes. In these studies, the authors aimed to determine whether morphine administered for a clinically relevant time period (7 days) in a tibia fracture orthopedic surgery model had adverse effects on postoperative recovery. METHODS: Mice were given morphine twice daily for 7 days after unilateral tibial fracture and intramedullary pin fixation to model orthopedic surgery and limb trauma. Mechanical allodynia, limb-specific weight bearing, gait changes, memory, and anxiety were measured after injury. In addition, spinal cord gene expression changes as well as glial activation were measured. Finally, the authors assessed the effects of a selective Toll-like receptor 4 antagonist, TAK-242, on nociceptive and functional changes after injury. RESULTS: Tibial fracture caused several weeks of mechanical nociceptive sensitization (F(1, 216) = 573.38, P < 0.001, fracture + vehicle vs. sham + vehicle, n = 10 per group), and this change was exacerbated by the perioperative administration of morphine (F(1, 216) = 71.61, P < 0.001, fracture + morphine vs. fracture + vehicle, n = 10 per group). In additional testing, injured limb weight bearing, gait, and object location memory were worse in morphine-treated fracture mice than in untreated fracture mice. Postfracture expression levels of several genes previously associated with opioid-induced hyperalgesia, including brain-derived neurotrophic factor and prodynorphin, were unchanged, but neuroinflammation involving Toll-like receptor 4 receptor-expressing microglia was observed (6.8 ± 1.5 [mean ± SD] cells per high-power field for fracture + vehicle vs. 12 ± 2.8 fracture + morphine, P < 0.001, n = 8 per /group). Treatment with a Toll-like receptor 4 antagonist TAK242 improved nociceptive sensitization for about 2 weeks in morphine-treated fracture mice (F(1, 198) = 73.36, P < 0.001, fracture + morphine + TAK242 vs. fracture + morphine, n = 10 per group). CONCLUSIONS: Morphine treatment beginning at the time of injury impairs nociceptive recovery and other outcomes. Measures preventing glial activation through Toll-like receptor 4 signaling may reduce the adverse consequences of postoperative opioid administration.

The Speech Arm Vision Eyes (SAVE) scale predicts large vessel occlusion stroke as well as more complicated scales.

INTRODUCTION: The Speech Arm Vision Eyes (SAVE) scale, a 4-item clinical scale emphasizing binary scoring and avoidance of nuanced examination distinctions, predicts LVOs with similar characteristics as more complex scales. METHODS: Receiver operating characteristic analyses of the prospective STOPStroke study assessed the ability of the SAVE scale and other published scales to predict LVO. We identified scale thresholds with positive likelihood ratios with 95% confidence intervals of ≥5.0 or negative likelihood ratios with 95% confidence intervals of ≤0.5. RESULTS: 735patients were studied. LVO prevalence was 33%. Area under the curve was 0.79 for SAVE, 0.82 for FAST-ED, 0.80 for mNIHSS and NIHSS, and lower for all other scales. SAVE=4, EMSA=6, mNIHSS≥10, NIHSS≥16, and RACE≥8 had positive likelihood ratios with 95% confidence intervals ≥5.0. SAVE≥2, CPSS≥2, C-STAT≥1, EMSA≥4, FAST-ED≥3, G-FAST≥3, mNIHSS≥6, NIHSS≥9, PASS≥1, RACE≥2, VAN=1, and 3I-SS≥1 had negative likelihood ratios with 95% confidence intervals ≤0.5. CONCLUSIONS: SAVE=4 performed similarly to more complex scales at predicting LVO. Other simplified scales did not have thresholds with positive likelihood ratios with 95% confidence intervals ≥5.0. Validation is need in a prehospital cohort of patients with suspected stroke.

Altered metabolic pathways regulate synovial inflammation in rheumatoid arthritis.

Rheumatoid arthritis is characterized by synovial proliferation, neovascularization and leucocyte extravasation leading to joint destruction and functional disability. The blood vessels in the inflamed synovium are highly dysregulated, resulting in poor delivery of oxygen; this, along with the increased metabolic demand of infiltrating immune cells and inflamed resident cells, results in the lack of key nutrients at the site of inflammation. In these adverse conditions synovial cells must adapt to generate sufficient energy to support their proliferation and activation status, and thus switch their cell metabolism from a resting regulatory state to a highly metabolically active state. This alters redox-sensitive signalling pathways and also results in the accumulation of metabolic intermediates which, in turn, can act as signalling molecules that further exacerbate the inflammatory response. The RA synovium is a multi-cellular tissue, and while many cell types interact to promote the inflammatory response, their metabolic requirements differ. Thus, understanding the complex interplay between hypoxia-induced signalling pathways, metabolic pathways and the inflammatory response will provide better insight into the underlying mechanisms of disease pathogenesis.

The p53-signaling pathway and colorectal cancer: Interactions between downstream p53 target genes and miRNAs.

INTRODUCTION: We examined expression of genes in the p53-signaling pathway. We determine if genes that have significantly different expression in carcinoma tissue compared to normal mucosa also have significantly differentially expressed miRNAs. We utilize a sample of 217 CRC cases. METHODS: We focused on fold change (FC) > 1.50 or <0.67 for genes and miRNAs, that were statistically significant after adjustment for multiple comparisons. We evaluated the linear association between the differential expression of miRNA and mRNA. miRNA:mRNA seed-region matches also were determined. RESULTS: Eleven dysregulated genes were associated with 37 dysregulated miRNAs; all were down-stream from the TP53 gene. MiR-150-5p (HR = 0.82) and miR-196b-5p (HR 0.73) significantly reduced the likelihood of dying from CRC when miRNA expression increased in rectal tumors. CONCLUSIONS: Our data suggest that activation of p53 from cellular stress, could target downstream genes that in turn could influence cell cycle arrest, apoptosis, and angiogenesis through mRNA:miRNA interactions.

Autoinflammatory and autoimmune contributions to complex regional pain syndrome.

Complex regional pain syndrome (CRPS) is a highly enigmatic syndrome typically developing after injury or surgery to a limb. Severe pain and disability are common among those with chronic forms of this condition. Accumulating evidence suggests that CRPS may involve both autoinflammatory and autoimmune components. In this review article, evidence for dysfunction of both the innate and adaptive immune systems in CRPS is presented. Findings from human studies in which cytokines and other inflammatory mediators were measured in the skin of affected limbs are discussed. Additional results from studies of mediator levels in animal models are evaluated in this context. Similarly, the evidence from human, animal, and translational studies of the production of autoantibodies and the potential targets of those antibodies is reviewed. Compelling evidence of autoinflammation in skin and muscle of the affected limb has been collected from CRPS patients and laboratory animals. Cytokines including IL-1ß, IL-6, TNFα, and others are reliably identified during the acute phases of the syndrome. More recently, autoimmune contributions have been suggested by the discovery of self-directed pain-promoting IgG and IgM antibodies in CRPS patients and model animals. Both the autoimmune and the autoinflammatory components of CRPS appear to be regulated by neuropeptide-containing peripheral nerve fibers and the sympathetic nervous system. While CRPS displays a complex neuroimmunological pathogenesis, therapeutic interventions could be designed targeting autoinflammation, autoimmunity, or the neural support for these phenomena.

Variables affecting penetrance of gastric and duodenal phenotype in familial adenomatous polyposis patients.

BACKGROUND: Patients with familial adenomatous polyposis (FAP) frequently undergo colectomy to reduce the 70 to 90% lifetime risk of colorectal cancer. After risk-reducing colectomy, duodenal cancer and complications from duodenal surgeries are the main cause of morbidity. Our objective was to prospectively describe the duodenal and gastric polyp phenotype in a cohort of 150 FAP patients undergoing pre-screening for a chemoprevention trial and analyze variables that may affect recommendations for surveillance. METHODS: Individuals with a diagnosis of FAP underwent prospective esophagogastroduodenoscopy using a uniform system of mapping of size and number of duodenal polyps for a 10 cm segment. Gastric polyps were recorded as the total number. RESULTS: The distribution of the count and sum diameter of duodenal polyps were statistically different in two genotype groups, those with APC mutations associated with classic FAP had a greater count (median 17) and sum diameter of polyps (median 32 mm) than those with APC mutations associated with attenuated FAP (median count 4 and median sum diameter of 7 mm) (p < 0.0001). The number of gastric polyps did not differ based on genotype (p = 0.67) but advancing age correlated with severity of gastric polyposis (p = 0.019). Spigelman (modified) staging of II or greater was found in 88% of classic FAP patients and 48% attenuated FAP patients. Examples of severe and mild upper GI phenotype are observed in patients with identical APC mutations, showing that the APC mutation location is not absolutely predictive of an upper GI phenotype. CONCLUSIONS: Most FAP patients have duodenal and gastric polyps which become more prevalent and advanced with age. Standard upper endoscopic surveillance is recommended based on personal history independent of APC mutation location. TRIAL REGISTRATION: NCT 01187901 registered August 24, 2010, prospective to enrollment.

The TGFß-signaling pathway and colorectal cancer: associations between dysregulated genes and miRNAs.

BACKGROUND: The TGFß-signaling pathway plays an important role in the pathogenesis of colorectal cancer (CRC). Loss of function of several genes within this pathway, such as bone morphogenetic proteins (BMPs) have been seen as key events in CRC progression. METHODS: In this study we comprehensively evaluate differential gene expression (RNASeq) of 81 genes in the TGFß-signaling pathway and evaluate how dysregulated genes are associated with miRNA expression (Agilent Human miRNA Microarray V19.0). We utilize paired carcinoma and normal tissue from 217 CRC cases. We evaluate the associations between differentially expressed genes and miRNAs and sex, age, disease stage, and survival months. RESULTS: Thirteen genes were significantly downregulated and 14 were significantly upregulated after considering fold change (FC) of > 1.50 or < 0.67 and multiple comparison adjustment. Bone morphogenetic protein genes BMP5, BMP6, and BMP2 and growth differentiation factor GDF7 were downregulated. BMP4, BMP7, INHBA (Inhibin beta A), TGFBR1, TGFB2, TGIF1, TGIF2, and TFDP1 were upregulated. In general, genes with the greatest dysregulation, such as BMP5 (FC 0.17, BMP6 (FC 0.25), BMP2 (FC 0.32), CDKN2B (FC 0.32), MYC (FC 3.70), BMP7 (FC 4.17), and INHBA (FC 9.34) showed dysregulation in the majority of the population (84.3, 77.4, 81.1, 80.2, 82.0, 51.2, and 75.1% respectively). Four genes, TGFBR2, ID4, ID1, and PITX2, were un-associated or slightly upregulated in microsatellite-stable (MSS) tumors while downregulated in microsatellite-unstable (MSI) tumors. Eight dysregulated genes were associated with miRNA differential expression. E2F5 and THBS1 were associated with one or two miRNAs; RBL1, TGFBR1, TGIF2, and INHBA were associated with seven or more miRNAs with multiple seed-region matches. Evaluation of the joint effects of mRNA:miRNA identified interactions that were stronger in more advanced disease stages and varied by survival months. CONCLUSION: These data support an interaction between miRNAs and genes in the TGFß-signaling pathway in association with CRC risk. These interactions are associated with unique clinical characteristics that may provide targets for further investigations.

Oxidative Stress Contributes to Fracture/Cast-Induced Inflammation and Pain in a Rat Model of Complex Regional Pain Syndrome.

Clinical evidence suggests that vitamin C (Vit C) may protect against the development of complex regional pain syndrome (CRPS) after fracture or surgery. Tibia fracture followed by 4 weeks of cast immobilization (fracture/cast) in rats results in nociceptive, vascular, and bone changes resembling clinical CRPS. In this study, fracture/cast rats were treated with the oxidative stress inhibitors Vit C, N-acetyl cysteine, or 4-hydroxy-2,2,6,6-tetramethylpiperidin-1-oxyl to examine their effects on CRPS-related nociceptive and vascular changes. Administration of these agents significantly reduced fracture/cast-induced cutaneous allodynia by 64 to 78%, muscle hyperalgesia by 34 to 40%, and hind limb unweighting by 48 to 89%. Treatments with Vit C and N-acetyl cysteine reduced the oxidative stress markers malondialdehyde in the skin, muscle, and sciatic nerve, and lactate in the gastrocnemius muscle of the fracture/cast limb. Furthermore, Vit C treatment inhibited the post-fracture upregulation of substance P and calcitonin gene-related peptide in the sciatic nerve and the increased expression of the pain-related inflammatory mediators, including interleukin (IL)-6, and nerve growth factor in the skin and IL-1ß, and IL-6 in the muscle of the post-fracture/cast limb. These data suggest that oxidative stress may contribute to the nociceptive features of the rat CRPS model. PERSPECTIVE: Vit C reduced the CRPS-like signs, oxidative stress, and the upregulation of neuropeptide production and inflammatory mediators observed after tibia fracture and casting in rats. Limiting oxidative stress by use of Vit C or alternative strategies could reduce the risk of developing CRPS after surgery or other forms of trauma.

The MAPK-Signaling Pathway in Colorectal Cancer: Dysregulated Genes and Their Association With MicroRNAs.

Mitogen-activated protein kinase (MAPK) pathways regulate many cellular functions including cell proliferation and apoptosis. We examined associations of differential gene and microRNA (miRNA) expression between carcinoma and paired normal mucosa for 241 genes in the KEGG-identified MAPK-signaling pathway among 217 colorectal cancer (CRC) cases. Gene expression data (RNA-Seq) and miRNA expression data (Agilent Human miRNA Microarray V19.0; Agilent Technologies Inc., Santa Clara, CA, USA) were analyzed. We first identified genes most strongly associated with CRC using a fold change (FC) of >1.50 or <0.67) that were statistically significant after adjustment for multiple comparisons. We then determined miRNAs associated with dysregulated genes and through miRNA:mRNA (messenger RNA) seed region matches discerned genes with a greater likelihood of having a direct biological association. Ninety-nine genes had a meaningful FC for all CRC, microsatellite unstable-specific tumors, or microsatellite stable-specific tumors. Thirteen dysregulated genes were associated with miRNAs, totaling 68 miRNA:mRNA associations. Thirteen of the miRNA:mRNA associations had seed region matches where the differential expression between the miRNA and mRNA was inversely related suggesting a direct association as a result of their binding. Several direct associations, upstream of ERK1/ERK2, JNK, and p38, were found for PDGFRA with 7 miRNAs; RASGRP3 and PRKCB with miR-203a; and TGFBR1 with miR-6071 and miR-2117. Other associations between miRNAs and mRNAs are most likely indirect, resulting from feedback and feed forward loops. Our results suggest that miRNAs may alter MAPK signaling through direct binding with key genes in this pathway. We encourage others to validate results in targeted CRC experiments that can help solidify important therapeutic targets.

Dysregulated genes and miRNAs in the apoptosis pathway in colorectal cancer patients.

Apoptosis is genetically regulated and involves intrinsic and extrinsic pathways. We examined 133 genes within these pathways to identify whether they are expressed differently in colorectal carcinoma (CRC) and normal tissue (N = 217) and if they are associated with similar differential miRNA expression. Gene expression data (RNA-Seq) and miRNA expression data (Agilent Human miRNA Microarray V19.0) were generated. We focused on dysregulated genes with a fold change (FC) of > 1.50 or < 0.67, that were significant after adjustment for multiple comparisons. miRNA:mRNA seed-region matches were determined. Twenty-three genes were significantly downregulated (FC < 0.67) and 18 were significantly upregulated (FC > 1.50). Of these 41 genes, 11 were significantly associated with miRNA differential expression. BIRC5 had the greatest number of miRNA associations (14) and the most miRNAs with a seed-region match (10). Four of these matches, miR-145-5p, miR-150-5p, miR-195-5p, and miR-650, had a negative beta coefficient. CSF2RB was associated with ten total miRNAs (five with a seed-region match, and one miRNA, miR-92a-3p, with a negative beta coefficient). Of the three miRNAs associated with CTSS, miR-20b-5p, and miR-501-3p, had a seed-region match and a negative beta coefficient between miRNA:mRNA pairs. Several miRNAs that were associated with dysregulated gene expression, seed-region matches, and negative beta coefficients also were associated with CRC-specific survival. Our data suggest that miRNAs could influence several apoptosis-related genes. BIRC5, CTSS, and CSF2R all had seed-region matches with miRNAs that would favor apoptosis. Our study identifies several miRNA associated with apoptosis-related genes, that if validated, could be important therapeutic targets.