Norman J. Galbraith

Blood-based microRNAs as biomarkers for the diagnosis of colorectal cancer: a systematic review and meta-analysis.

Background:Colorectal cancer (CRC) is common and associated with significant mortality. Current screening methods for CRC lack patient compliance. microRNAs (miRNAs), identified in body fluids, are negative regulators of gene expression and are dysregulated in many cancers, including CRC. This paper summarises studies identifying blood-based miRNAs dysregulated in CRC compared with healthy controls in an attempt to evaluate their use as a screening tool for the diagnosis of CRC.Methods:A search of electronic databases (PubMed and EMBASE) and grey literature was performed between January 2002 and April 2016. Studies reporting plasma or serum miRNAs in the diagnosis of CRC compared with healthy controls were selected. Patient demographics, type of patient sample (serum or plasma), method of miRNA detection, type of normalisation, and the number of significantly dysregulated miRNAs identified were recorded. Statistical evaluation of dysregulated miRNAs using sensitivity, specificity, and area under the curve (AUC) was performed.Results:Thirty-four studies investigating plasma or serum miRNAs in the diagnosis of CRC were included. A total of 31 miRNAs were found to be either upregulated (n=17) or downregulated (n=14) in CRC cases as compared with controls. Fourteen studies identified panels of ⩾2 dysregulated miRNAs. The highest AUC, 0.943, was identified using a panel of 4 miRNAs with 83.3% sensitivity and 93.1% specificity. Meta-analysis of studies identifying a single dysregulated miRNA in CRC cases compared with controls was performed. Overall sensitivity and specificity of 28 individual miRNAs in the diagnosis of CRC were 76% (95% CI 72%–80%) and 76% (95% CI 72%–80%), respectively, indicating good discriminative ability of miRNAs as biomarkers for CRC. These data did not change with sensitivity analyses.Conclusions:Blood-based miRNAs distinguish patients with CRC from healthy controls with high sensitivity and specificity comparable to other common and invasive currently used screening methods for CRC. In future, miRNAs may be used as a relatively non-invasive blood-based marker for detection of CRC.

A Highly Predictive Model for Diagnosis of Colorectal Neoplasms Using Plasma MicroRNA: Improving Specificity and Sensitivity.

Objective: To develop a plasma-based microRNA (miRNA) diagnostic assay specific for colorectal neoplasms, building upon our prior work. Background: Colorectal neoplasms [colorectal cancer (CRC) and colorectal advanced adenoma (CAA)] frequently develop in individuals at ages when other common cancers also occur. Current screening methods lack sensitivity, specificity, and have poor patient compliance. Methods: Plasma was screened for 380 miRNAs using microfluidic array technology from a “Training” cohort of 60 patients, (10 each) control, CRC, CAA, breast cancer, pancreatic cancer, and lung cancer. We identified uniquely dysregulated miRNAs specific for colorectal neoplasia (P < 0.05, false discovery rate: 5%, adjusted &agr; = 0.0038). These miRNAs were evaluated using single assays in a “Test” cohort of 120 patients. A mathematical model was developed to predict blinded sample identity in a 150 patient “Validation” cohort using repeat-sub-sampling validation of the testing dataset with 1000 iterations each to assess model detection accuracy. Results: Seven miRNAs (miR-21, miR-29c, miR-122, miR-192, miR-346, miR-372, and miR-374a) were selected based upon P value, area under the curve (AUC), fold change, and biological plausibility. Area under the curve (±95% confidence interval) for “Test” cohort comparisons were 0.91 (0.85–0.96) between all neoplasia and controls, 0.79 (0.70–0.88) between colorectal neoplasia and other cancers, and 0.98 (0.96–1.0) between CRC and colorectal adenomas. In our “Validation” cohort, our mathematical model predicted blinded sample identity with 69% to 77% accuracy, 67% to 76% accuracy, and 86% to 90% accuracy for each comparison, respectively. Conclusions: Our plasma miRNA assay and prediction model differentiate colorectal neoplasia from patients with other neoplasms and from controls with higher sensitivity and specificity compared with current clinical standards.

Rapid tissue regeneration induced by intracellular ATP delivery—A preliminary mechanistic study

We have reported a new phenomenon in acute wound healing following the use of intracellular ATP delivery—extremely rapid tissue regeneration, which starts less than 24 h after surgery, and is accompanied by massive macrophage trafficking, in situ proliferation, and direct collagen production. This unusual process bypasses the formation of the traditional provisional extracellular matrix and significantly shortens the wound healing process. Although macrophages/monocytes are known to play a critical role in the initiation and progression of wound healing, their in situ proliferation and direct collagen production in wound healing have never been reported previously. We have explored these two very specific pathways during wound healing, while excluding confounding factors in the in vivo environment by analyzing wound samples and performing in vitro studies. The use of immunohistochemical studies enabled the detection of in situ macrophage proliferation in ATP-vesicle treated wounds. Primary human macrophages and Raw 264.7 cells were used for an in vitro study involving treatment with ATP vesicles, free Mg-ATP alone, lipid vesicles alone, Regranex, or culture medium. Collagen type 1α 1, MCP-1, IL-6, and IL-10 levels were determined by ELISA of the culture supernatant. The intracellular collagen type 1α1 localization was determined with immunocytochemistry. ATP-vesicle treated wounds showed high immunoreactivity towards BrdU and PCNA antigens, indicating in situ proliferation. Most of the cultured macrophages treated with ATP-vesicles maintained their classic phenotype and expressed high levels of collagen type 1α1 for a longer duration than was observed with cells treated with Regranex. These studies provide the first clear evidence of in situ macrophage proliferation and direct collagen production during wound healing. These findings provide part of the explanation for the extremely rapid tissue regeneration, and this treatment may hold promise for acute and chronic wound care.

IκK-16 decreases miRNA-155 expression and attenuates the human monocyte inflammatory response

Excessive inflammatory responses in the surgical patient may result in cellular hypo-responsiveness, which is associated with an increased risk of secondary infection and death. microRNAs (miRNAs), such as miR-155, are powerful regulators of inflammatory signalling pathways including nuclear factor κB (NFκB). Our objective was to determine the effect of IκK-16, a selective blocker of inhibitor of kappa-B kinase (IκK), on miRNA expression and the monocyte inflammatory response. In a model of endotoxin tolerance using primary human monocytes, impaired monocytes had decreased p65 expression with suppressed TNF-α and IL-10 production (P < 0.05). miR-155 and miR-138 levels were significantly upregulated at 17 h in the impaired monocyte (P < 0.05). Notably, IκK-16 decreased miR-155 expression with a corresponding dose-dependent decrease in TNF-α and IL-10 production (P < 0.05), and impaired monocyte function was associated with increased miR-155 and miR-138 expression. In the context of IκK-16 inhibition, miR-155 mimics increased TNF-α production, while miR-155 antagomirs decreased both TNF-α and IL-10 production. These data demonstrate that IκK-16 treatment attenuates the monocyte inflammatory response, which may occur through a miR-155-mediated mechanism, and that IκK-16 is a promising approach to limit the magnitude of an excessive innate inflammatory response to LPS.

Temporal expression of circulating miRNA after severe injury

Background: Severe injury can lead to immune dysfunction and predispose patients to infection and death. Micro‐RNAs regulate gene expression and may act as biomarkers for susceptibility to infection. The aim of this study was to examine the temporal and differential expression of previously identified dysregulated micro‐RNAs in patients with severe injury. Methods: Fourteen severely injured patients requiring transfusion were enrolled prospectively in this study approved by our institutional review board. Inclusion criteria consisted of adult patients deemed clinically to be in hemorrhagic shock necessitating transfusion in the acute phase of their injury care. Peripheral blood samples were obtained after admission to the surgical intensive care unit and again at 6, 12, 24, and 48 hours after admission. The samples obtained at arrival to the intensive care unit and 24 and 48 hours later were analyzed in this data set. Fourteen healthy volunteers served as controls. The 10 dysregulated micro‐RNAs identified in a prior study at the 12‐hour time point and important genes in innate immunity were measured using quantitative reverse transcription–polymerase chain reaction. Results: The participants were 21–77 years old (median, 42), 78% were male, and their Injury Severity Score ranged from 11 to 43 (median, 27); 11 had blunt and 3 had penetrating injuries. Three were intubated and 5 had received blood products before arrival at the hospital. Base deficit on hospital admission was 3–20 (median, 9). All patients required blood transfusion secondary to blood loss sustained during injury. Eleven of the 14 patients went directly to the operating room from the emergency department for control of the source of hemorrhage. Survival to discharge was 93%. Seven patients developed infection. Compared with healthy controls, miR‐106a was downregulated at all time points compared with controls (P < .05). miR‐618 was upregulated in initial blood draws (P < .05) and at 24 and 48 hours (P < .06). Tumor necrosis factor &agr; and human leukocyte antigen‐DR (HLA‐DR) were downregulated, and interleukin‐10 and PD‐L1 were upregulated (P < .05). In patients who developed infection, miR‐106a levels appeared more downregulated than those who did not develop infection. Conclusion: miR‐106a was downregulated in trauma patients after major injury for up to 48 hours after intensive care unit admission. Tumor necrosis factor &agr; and interleukin‐10 are targeted by miR‐106a, which are regulators of the immune response. Manipulation of micro‐RNA expression may be a therapeutic target for immune dysfunction.

Long-term outcomes following ileal pouch-anal anastomosis in patients with indeterminate colitis

Background. The advisability of performing ileal pouch–anal anastomosis for patients with indeterminate colitis is debated. Indeterminate colitis is found in up to 15% of inflammatory bowel disease colectomy specimens. We determined long‐term outcomes in patients diagnosed with indeterminate colitis undergoing ileal pouch–anal anastomosis. Methods. Fifty‐six patients were included with a mean follow‐up of 14 ± 7 years. Long‐term behavior was defined based on surgeon assessment as “Crohn disease–like” in patients who subsequently developed clear signs of Crohn disease and as “non‐Crohn disease–like.” Long‐term function was assessed using the Cleveland Global Quality of Life and Pouch Functional Score. Results. Thirty‐nine percent of patients developed Crohn disease–like behavior, and 61% developed non‐Crohn disease–like behavior. Both groups experienced a high rate of pouchitis (57%). Crohn disease–like patients required more anti‐inflammatory/immunomodulatory medications (95% vs 18%, P < .001), dilatations for afferent‐limb strictures (41% vs 0%, P < .001), and pouch reoperations (32% vs 6%, P = .02). Eight patients required pouch excision or diversion (7 with Crohn disease–like behavior). The Pouch Functional Score was equivalent between groups. Conclusion. Long‐term function after ileal pouch–anal anastomosis for the majority of indeterminate colitis patients was good. Approximately 40% eventually exhibited Crohn disease–like behavior, but the majority had acceptable function and quality of life. Ileal pouch–anal anastomosis is an appropriate surgical option for indeterminate colitis patients after informed consent.

Plasma microRNA Profile Differentiates Crohn’s Colitis From Ulcerative Colitis

Abstract Background Inflammatory bowel disease (IBD) is commonly divided into 2 entities: Crohn’s disease (CD) and ulcerative colitis (UC). Differentiating between these entities when dealing with IBD confined to the colon is important, especially when planning surgical treatment. Due to ambiguous histological or endoscopic findings, accurate diagnosis is not possible in up to 15% of cases. The aim of this study was to determine whether plasma microRNAs (miRNAs) can help differentiate Crohn’s colitis (CC) from ulcerative colitis. Methods Patients with isolated CC and with UC were enrolled in our study from January 2010 to May 2016. Peripheral blood was collected, and total RNA was isolated from plasma. Screening was performed for 380 common miRNAs. miRNAs that were differentially expressed between these 2 groups were chosen, and their differential expression was confirmed using single miRNA assays in a larger sample size. A predictive model was generated using these data. Significantly differentially expressed miRNAs were then validated utilizing the predictive model to assess blinded data from the single assays. Results Screening was performed on 8 patients from each group. Seven differentially expressed miRNAs were chosen for single assay confirmation. Two miRNAs (miR-598, miR-642) were consistently different between the patient groups (P = 0.013, P = 0.005). Using blinded data, these 2 miRNAs were validated using the predictive model, achieving an overall accuracy of 75% (95% confidence interval, 40.7–92.9). Conclusions We identified 2 plasma miRNAs that differentiated CC from UC. Our data indicate the promise and feasibility of a plasma miRNA–based assay to distinguish between these 2 conditions.

The effect of IκK-16 on lipopolysaccharide-induced impaired monocytes

This study focuses on impaired monocyte function, which occurs in some patients after trauma, major elective surgery, or sepsis. This monocyte impairment increases the risk of secondary infection and death. We aimed to determine the influence IκK-16 had on monocytes using an ex-vivo model of human monocyte impairment. We included the effects of the well-studied comparators interferon-gamma (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) on impaired monocytes. Primary human monocytes were stimulated with 10ng/mL of lipopolysaccharide (LPS) for 16h and then challenged with 100ng/mL LPS to assess the monocyte inflammatory response. Treatment regimens, consisting of either IκK-16, IFN-γ, or GM-CSF, were administered to impaired monocytes near the time of initial LPS stimulation. Stimulation with 10ng/mL LPS initially promoted a pro-inflammatory response but subsequently impaired production of both tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) and decreased HLA-DR expression. IκK-16 treatment attenuated TNF-α production and programmed death-ligand 1 (PD-L1) expression and increased IL-10 and CD14 expression. IFN-γ treatment increased TNF-α production as well as PD-L1 and HLA-DR expression. In conclusion, limiting early inflammation with IκK-16 suppresses TNF-α production and PD-L1 expression but enhances IL-10 production and preserves CD14 expression for potential future exposure to infective stimuli.