Myeloperoxidase-positive Cells Research Articles

Incontinence-Associated Dermatitis-Like Skin Changes Induced by the Application of Absorbent Pads Containing Bacteria and Artificial Urine in Rats.

Incontinence-associated dermatitis (IAD) is one of the most serious complications in older people with incontinence. Controlling urine property in absorbent pads could be effective for preventing IAD caused by bacterial urine. However, no animal model has been established to evaluate their effectiveness. This study aimed to induce IAD-like skin changes using absorbent pads containing bacterial urine and to confirm their pathophysiology in rats. Hairless Wistar Yagi rats were divided into the bacteria-containing urine (BU) and the bacteria-free urine (U) groups. A 10-h-attachment of absorbent pads containing artificial urine with/without bacteria to the skin pretreated with sodium lauryl sulfate was performed repeatedly for 5 days. Macroscopic findings and skin barrier function were examined every day, and histological changes, inflammatory responses and bacterial quantification in tissue samples were examined on Day 5. The BU group exhibited significant skin redness from Day 3, significant elevation of transepidermal water loss from Day 1, and histological changes, including significantly thickened epidermis, abnormal keratinocyte differentiation and erythrocyte leakage. Inflammation, confirmed by higher myeloperoxidase-positive cells, elevated tumour necrosis factor-α expression, and vascular endothelial damage, indicated by CD31 and pentraxin 3-positive cells, were observed in the BU group. The bacterial quantification showed no significant difference between the groups. IAD-like skin changes including histological changes and inflammation were suggested to be caused by urine properties altered by bacteria. This study proposed a new animal model for evaluating the effectiveness of absorbent pads in controlling the urine properties of bacterial urine on preventing IAD.

Spontaneous Lethal Outbreak of Influenza A Virus Infection in Vaccinated Sows on Two Farms Suggesting the Occurrence of Vaccine-Associated Enhanced Respiratory Disease with Eosinophilic Lung Pathology.

Influenza A virus (IAV) infections in swine are usually subclinical, but they can reach high morbidity rates. The mortality rate is normally low. In this study, six vaccinated, spontaneously deceased sows revealed IAV infection and enhanced neutrophilic bronchopneumonia with unexpectedly large numbers of infiltrating eosinophils. The purpose of this study was to characterize these lung lesions with special emphasis on the phenotypes of inflammatory cells, the presence of eosinophilic peroxidase (EPO), and neutrophil extracellular traps (NETs). The number of Sirius red-stained eosinophils was significantly higher in the lungs of IAV-infected sows compared to healthy pigs, indicating a migration of eosinophils from blood vessels into the lung tissue stimulated by IAV infection. The detection of intra- and extracellular EPO in the lungs suggests its contribution to pulmonary damage. The presence of CD3+ T lymphocytes, CD20+ B lymphocytes, and Iba-1+ macrophages indicates the involvement of cell-mediated immune responses in disease progression. Furthermore, high numbers of myeloperoxidase-positive cells were detected. However, DNA-histone-1 complexes were reduced in IAV-infected sows, leading to the hypothesis that NETs are not formed in the IAV-infected sows. In conclusion, our findings in the lungs of IAV-infected vaccinated sows suggest the presence of so far unreported field cases of vaccine-associated enhanced respiratory disease.

Identification and characterization of myeloid cells localized in the tadpole liver cortex in Xenopus laevis

In the present study, using transgenic frogs that express GFP specifically in myeloid cells under the myeloperoxidase enhancer sequence, we found that myeloperoxidase-positive cells are localized in the liver cortex at the late tadpole stages. Immunohistochemical analysis revealed that myelopoiesis in the liver cortex became evident after st. 50 and reached its peak by st. 56. Transplantation experiments indicated that cells with a high density at the liver cortex were derived from the dorso-lateral plate tissue in the neurula embryo. Analysis of smear samples of the cells isolated from collagenase-treated liver tissues of the transgenic tadpoles indicated that myeloid cells were the major population of blood cells in the larval liver and that, in addition to myeloid colonies, erythroid colonies expanded in entire liver after metamorphosis. Cells that were purified from the livers of transgenic tadpoles according to the GFP expression exhibited the multi-lobed nuclei. The results of present study provide evidence that the liver cortex of the Xenopus tadpole is a major site of granulopoiesis.

Picalm/MLLT10 Expression in Zebrafish Is Associated with an Increase of Hematopoietic Stem Cells and Leads to Leukemia

The PICALM/ MLLT10 (C/A)fusion gene is the result of the (10;11)(p13;q14) translocation, and is found in about 1% of all AML patients, 30% of T-cell ALL with a TCR γ/∂ rearrangement and in some patients with lymphoma. AML patients with a t(10;11)(p13;q14) have poor long-term survival. Mouse models of t(10;11)(p13;q14) show that C/A and the C/A minimal fusion (CA/MF) are strong oncogenic drivers, and that C/A must be expressed in the hematopoietic stem cells (HSCs) to cause leukemia. Patient samples and mouse models of C/A leukemia show that these leukemias harbor additional, cooperating mutations. Zebrafish (ZF) have become an attractive animal model for studying hematological disorders. ZF can be easily genetically modified and kept in large numbers. Although T cell ALL models have been successful in ZF, AML models have not been as successful. Most ZF AML models fail to recapitulate the phenotype, especially transplantability. Here we report a C/A transgenic zebrafish AML model (R:CA/MF). We used the murine Runx1 promoter to direct CA/MF expression to zebrafish HSCs using Tol2-mediated transgenesis. We detected the expression of the CA/MF transcript using RT-PCR in 3 dpf (days post fertilisation) heart marker positive transgenic F0 embryos and the expression of FLAG-tagged CA/MF protein with whole mount in situ immuno histochemistry in stable transgenic R:CA/MF embryos at 3 dpf. We performed whole mount in situ hybridisation (WMISH) and qPCR for hematopoietic genes to examine the impact of CA/MF on zebrafish hematopoiesis. Based on qPCR analysis, we observed that the expression of pu1 (p value <0.0120) and gata1 (p value <0.0004) was significantly decreased in 3 dpf R:CA/MF embryos compared to WT, whereas the expression of mpx at 2 dpf (p value <0.0132) and 3 dpf (p value <0.0378) was significantly increased. Our WMISH analysis showed that the expression of lyz was significantly decreased in R:CA/MF compared to WT embryos at 2 dpf. To investigate the effect of CA/MF expression on HSCs, we injected the R:CA/MF construct into fertilised oocytes of R:GFP;kdrl:RFP fish (R:GFP marks HSCs and Kdrl:RFP marks blood vessels). The results from intravital confocal microscopy showed that the 3 dpf R:GFP larvae that had been injected with the R:CA/MF vector had about a 2.5 fold increase (p value <0.0001) of the GFP expressing cells (HSCs) in both the caudal hematopoietic tissue and circulation compared to control (R:Gal4) and un-injected larvae. These results were also confirmed by flow cytometry. CA/MF transgenic zebrafish developed an aggressive hematological malignancy with a latency of about 1 year and 60 to 70% penetrance. 68% of F1 R:CA/MF (15/22), 64% of F2 R:CA/MF (45/70), 62% of F3 R:CA/MF (22/35) and 62% of F4 R:CA/MF (25/40) adult ZF were euthanised or found dead. The median survival for F1 CA/MF transgenic ZF was 65.6 weeks and 69, 78.4, and 79.1 weeks for F2, F3, and F4, respectively. Histology analysis of R:CA/MF leukemic fish showed hypercellularity in kidney, spleen, heart, gills and eyes. Myeloperoxidase (MPO) staining showed that the number of MPO positive cells in kidney marrow was about 50% higher (p value <0.0001) in R:CA/MF compared to WT. Peripheral blood film analysis of R:CA/MF showed the presence of blast-like cells. In addition, R:CA/MF malignancies were associated with an expansion of either the precursor compartment (N=20/31), the myeloid compartment (N=8/31) or the lymphoid compartment (N=3/31) on forward/side scatter flow cytometry analysis. Transplantation assays showed that R:CA/MF leukemias were serially transplantable. The latency to leukemia development was about six weeks in the first, second, and third recipient fish, which was much shorter than the latency for the primary R:CA/MF leukemias. Our preliminary whole-exome sequencing (WES) analysis identified somatically mutated genes in CA/MF zebrafish leukemia with some of these genes having human orthologues that are known to be somatically mutated in human leukemia suggesting that these are cooperating driver mutations. Preliminary RNA-Seq experiments revealed that genes in the KRAS and MTOR pathways had increased expression in the CA/MF ZF leukemia. R:CA/MF leukemia model is a valuable model for understanding the molecular mechanisms of malignant cell transformation of C/A. It allows us to investigate the effects of C/A on HSCs in vivo and to study the fully developed leukemia via serial transplantation, WES, and RNA-Seq.

Intragastric infusion of a liquid diet with low-methoxyl pectin alleviates fecal inconsistency and local proinflammatory cytokine expression in lipopolysaccharide-septic rats

ObjectiveDiarrhea interrupts enteral nutrition management in hospitalized patients with severe illnesses, such as sepsis. Pectin, a water-soluble dietary fiber, has the potential to maintain intestinal function and may reduce inflammatory reactions. The aim of this study was to demonstrate that the addition of low-methoxyl (LM) pectin to a liquid diet suppresses softening of stool texture and reduces tissue inflammatory responses in enteral nutrition management during sepsis. MethodsA fat-enriched liquid diet with LM pectin (P-EN) or a liquid diet without dietary fiber (FF-EN) was given continuously to rats through a gastric catheter. Lipopolysaccharide (LPS; 10 mg/kg) was injected intraperitoneally 24 h (study 1) and 7 h (study 2) before sacrifice. ResultsLPS injection significantly worsened fecal property scores in rats infused with FF-EN compared with the rats given P-EN in study 1. Whereas many myeloperoxidase-positive cells infiltrated the liver, and the hepatic expressions of chemokine genes were markedly elevated 24 h after LPS administration, these findings were clearly alleviated in the LM pectin-containing liquid diet group. In study 2, protein expressions of proinflammatory cytokines, such as small intestinal tumor necrosis factor-α and hepatic interleukin-1β, and interleukin-6, were significantly downregulated in the P-EN LPS group compared with the FF-EN LPS group. ConclusionsA liquid diet containing LM pectin allows enteral nutrition management with a low risk for diarrhea and reduces local inflammation under septic conditions.

Effects of multiple pro-inflammatory stimuli in utero on the ileum of extremely premature ovine fetuses.

Chorioamnionitis is common in preterm birth and associated with a higher risk of intestinal inflammation and necrotizing enterocolitis. The intestinal inflammation influences the enteric nervous system development. We hypothesized that inflammation and innervation in the fetal ileum may be modified by chorioamnionitis induced by repeated challenge with lipopolysaccharide and/or preexisting Ureaplasma parvum infection at very low gestational age equivalent to 60% of term. Time mated ovine fetuses were exposed by intraamniotic injections to chronic Ureaplasma parvum for 24 days and/or lipopolysaccharide for 7 days, 2 days, or 7 & 2 days before delivery at 94 +/-2 days of gestational age (term at approximately 150 days). Intestinal inflammation as well as structural changes of the enteric nervous system were assessed. Lipopolysaccharide exposure increased CD3 and myeloperoxidase-positive cells (p < 0.05). Repetitive exposure to lipopolysaccharide or combined Ureaplasma parvum & lipopolysaccharide exposure increased intestinal inflammation (p < 0.05). The reduction of nuclei of neurons was most significant with repetitive lipopolysaccharide exposures but could be detected in all other intervention groups compared to the control group. Astrocyte-like glial cells increased if exposure to lipopolysaccharide was only 2 days before delivery or chronic exposure to Ureaplasma parvum existed beforehand (p < 0.05). After exposure to chorioamnionitis induced by Ureaplasma parvum and/or lipopolysaccharide, inflammatory responses as well as structural changes of the enteric nervous system were more pronounced the longer and the more frequent the exposure to pro-inflammatory stimuli before birth. These changes may cause functional effects of clinical importance.

COMBINATION THERAPY WITH A SENSE OLIGONUCLEOTIDE TO INDUCIBLE NITRIC OXIDE SYNTHASE MRNA AND HUMAN SOLUBLE THROMBOMODULIN IMPROVES SURVIVAL OF SEPSIS MODEL RATS AFTER PARTIAL HEPATECTOMY.

Sepsis after a major hepatectomy is a critical problem. In septic shock, the inflammatory mediator, nitric oxide (NO), is overproduced in hepatocytes and macrophages. The natural antisense (AS) transcripts, non-coding RNAs, are transcribed from a gene that encodes inducible nitric oxide synthase (iNOS). iNOS AS transcripts interact with and stabilize iNOS mRNAs. A single-stranded "sense oligonucleotide" (designated as SO1) corresponding to the iNOS mRNA sequence inhibits mRNA-AS transcript interactions and reduces iNOS mRNA levels in rat hepatocytes. In contrast, recombinant human soluble thrombomodulin (rTM) treats disseminated intravascular coagulopathy by suppressing coagulation, inflammation, and apoptosis. In this study, the combination therapy of SO1 and a low dose of rTM was evaluated for hepatoprotection in a rat septic shock model after partial hepatectomy. Rats underwent 70% hepatectomy, followed by intravenous (i.v.) injection of lipopolysaccharide (LPS) after 48 h. SO1 was injected (i.v.) simultaneously with LPS, whereas rTM was injected (i.v.) 1 h before LPS injection. Similarly to our previous report, SO1 increased survival after LPS injection. When rTM, which has different mechanisms of action, was combined with SO1, it did not interfere with the effect of SO1 and showed a significant increase in survival compared with LPS alone treatment. In serum, the combined treatment decreased NO levels. In the liver, the combined treatment inhibited iNOS mRNA and protein expression. A decreased iNOS AS transcript expression by the combined treatment was also observed. The combined treatment decreased mRNA expression of the inflammatory and pro-apoptotic genes while increasing that of the anti-apoptotic gene. Furthermore, the combined treatment reduced the number of myeloperoxidase-positive cells. These results suggested that the combination of SO1 and rTM has therapeutic potential for sepsis.

Endothelial cells overexpressing CXCR1/2 are renoprotective in rats with acute kidney injury.

Inflammation that develops with the release of chemokines and cytokines during acute kidney injury (AKI) has been shown to participate in functional renal recovery. Although a major research focus has been on the role of macrophages, the family of C-X-C motif chemokines that promote neutrophil adherence and activation also increases with kidney ischemia-reperfusion (I/R) injury. This study tested the hypothesis that intravenous delivery of endothelial cells (ECs) that overexpress (C-X-C motif) chemokine receptors 1 and 2 (CXCR1 and CXCR2, respectively) improves outcomes in kidney I/R injury. Overexpression of CXCR1/2 enhanced homing of endothelial cells to I/R-injured kidneys and limited interstitial fibrosis, capillary rarefaction, and tissue injury biomarkers (serum creatinine concentration and urinary kidney injury molecule-1) following AKI and also reduced expression of P-selectin and the rodent (C-X-C motif) chemokine cytokine-induced neutrophil chemoattractant (CINC)-2β as well as the number of myeloperoxidase-positive cells in the postischemic kidney. The serum chemokine/cytokine profile, including CINC-1, showed similar reductions. These findings were not observed in rats given endothelial cells transduced with an empty adenoviral vector (null-ECs) or a vehicle alone. These data indicate that extrarenal endothelial cells that overexpress CXCR1 and CXCR2, but not null-ECs or vehicle alone, reduce I/R kidney injury and preserve kidney function in a rat model of AKI.NEW & NOTEWORTHY Inflammation facilitates kidney ischemia-reperfusion (I/R) injury. Endothelial cells (ECs) that were modified to overexpress (C-X-C motif) chemokine receptor (CXCR)1/2 (CXCR1/2-ECs) were injected immediately following kidney I/R injury. The interaction of CXCR1/2-ECs, but not ECs transduced with an empty adenoviral vector, with injured kidney tissue preserved kidney function and reduced production of inflammatory markers, capillary rarefaction, and interstitial fibrosis. The study highlights a functional role for the C-X-C chemokine pathway in kidney damage following I/R injury.

Molecular Magnetic Resonance Imaging of Aneurysmal Inflammation Using a Redox Active Iron Complex.

Inflammation plays a key role in driving brain aneurysmal instability and rupture, but clinical tools to noninvasively differentiate between inflamed and stable aneurysms are lacking. We hypothesize that imaging oxidative changes in the aneurysmal microenvironment driven by myeloid inflammatory cells may represent a noninvasive biomarker to evaluate rupture risk. In this study, we performed initial evaluation of the oxidatively activated probe Fe-PyC3A as a tool for magnetic resonance imaging (MRI) of inflammation in a rabbit model of saccular aneurysm. The difference in longitudinal relaxivity ( r1 ) in reduced and oxidized states of Fe-PyC3A was measured in water and blood plasma phantoms at 3 T. A rabbit saccular aneurysm model was created by endovascular intervention/elastinolysis with subsequent decellularization in situ. Rabbits were imaged at 4 weeks (n = 4) or 12 weeks (n = 4) after aneurysmal induction, when luminal levels of inflammation reflected by the presence of myeloperoxidase positive cells are relatively high and low, respectively, using a 3 T clinical scanner. Both groups were imaged dynamically using a 2-dimensional T1-weighted fast field echo pulse MRI sequence before and up to 4 minutes postinjection of Fe-PyC3A. Dynamic imaging was then repeated after an injection of gadobutrol (0.1 mmol/kg) as negative control probe. Rabbits from the 12-week aneurysm group were also imaged before and 20 minutes and 3 hours after injection of Fe-PyC3A using an axial respiratory gated turbo-spin echo (TSE) pulse sequence with motion-sensitized driven equilibrium (MSDE) preparation. The MSDE/TSE imaging was repeated before, immediately after dynamic acquisition (20 minutes postinjection), and 3 hours after injection of gadobutrol. Aneurysmal enhancement ratios (ERs) were calculated by dividing the postinjection aneurysm versus skeletal muscle contrast ratio by the preinjection contrast ratio. After imaging, the aneurysms were excised and inflammatory infiltrate was characterized by fluorometric detection of myeloperoxidase activity and calprotectin immunostaining, respectively. In vitro relaxometry showed that oxidation of Fe-PyC3A by hydrogen peroxide resulted in a 15-fold increase of r1 at 3 T. Relaxometry in the presence of blood plasma showed no more than a 10% increase of r1 , indicating the absence of strong interaction of Fe-PyC3A with plasma proteins. Dynamic imaging with Fe-PyC3A generated little signal enhancement within the blood pool or adjacent muscle but did generate a transient increase in aneurysmal ER that was significantly greater 4 weeks versus 12 weeks after aneurysm induction (1.6 ± 0.30 vs 1.2 ± 0.03, P < 0.05). Dynamic imaging with gadobutrol generated strong aneurysmal enhancement, but also strong enhancement of the blood and muscle resulting in smaller relative ER change. In the 12-week group of rabbits, MSDE/TSE imaging showed that ER values measured immediately after dynamic MRI (20 minutes postinjection) were significantly higher ( P < 0.05) in the case of Fe-PyC3A (1.25 ± 0.06) than for gadobutrol injection (1.03 ± 0.03). Immunohistochemical corroboration using anticalprotectin antibody showed that leukocyte infiltration into the vessel walls and luminal thrombi was significantly higher in the 4-week group versus 12-week aneurysms (123 ± 37 vs 18 ± 7 cells/mm 2 , P < 0.05). Magnetic resonance imaging using Fe-PyC3A injection in dynamic or delayed acquisition modes was shown to generate a higher magnetic resonance signal enhancement in aneurysms that exhibit higher degree of inflammation. The results of our pilot experiments support further evaluation of MRI using Fe-PyC3A as a noninvasive marker of aneurysmal inflammation.

A CASE OF RECURRENT HISTIOCYTOID SWEET SYNDROME

<h3>Introduction</h3> Sweet syndrome (SS) is an acute febrile neutrophilic dermatosis characterized by abrupt onset of painful plaques, nodules, or papules, which can be associated with malignancies, auto-immune diseases, infections, or drug exposures. Identifying triggers, if any, especially in the context of rare life-threatening histological variants, is crucial for disease management. <h3>Case Description</h3> Ms. S is a 44-year-old woman who presented for evaluation of a 9-year history of erythematous, painful, non-pruritic, indurated plaques on her abdomen and thighs that can last 1-2 months, leaving residual bruising, often associated with fevers, headaches, pleuritic chest pain, dyspnea, and fatigue. Triggers include intense workouts and elevated stress. Lesions initially occurred once every 2 years, but now occur bi-annually. Systemic steroids helped the most, while colchicine and Benadryl provided marginal relief. Rheumatological, immunodeficiency, infectious, malignancy and autoinflammatory disease work-up was unremarkable. Biopsy with direct immunofluorescence revealed histopathologic features consistent with histiocytoid SS. Given infrequency of symptoms, we decided against chronic suppressive therapy with steroids. <h3>Discussion</h3> Histiocytoid SS is a rare histological variant of neutrophilic SS, with dermal infiltrate mainly composed of lymphocytes and histiocytoid myeloperoxidase-positive cells, which may be associated with a higher risk of hematologic malignancy. This case demonstrates the extensive work-up required to evaluate for underlying diseases, as treatment of the underlying condition can often mitigate these lesions. We were unable to identify any triggers, and she presented with a rare histological variant not well described in the literature, making management more challenging. Continued surveillance, particularly for malignancy, is warranted given that this rash can potentially precede cancer.

Ticagrelor versus clopidogrel in reducing inflammatory cell infiltration of thrombus aspirated in patients with ST-elevation myocardial infarction.

Ticagrelor provides more rapid, potent, and consistent anti-platelet efficacy than clopidogrel. This randomized trial aimed to evaluate the anti-inflammation effects of ticagrelor versus clopidogrel on thrombus aspirated from the ST-elevation myocardial infarction (STEMI) patients. A total of 98 patients with STEMI and intended percutaneous coronary intervention (PCI) were randomly assigned to receive clopidogrel (600-mg loading dose) or ticagrelor (180-mg loading dose), of whom 55 with large thrombus burden underwent thrombus aspiration during PCI. Thrombus specimens were successfully aspirated from 49 patients. Finally, 24 patients in the clopidogrel group and 23 in the ticagrelor group completed the study. Inflammatory cells within thrombi were assessed by hematoxylin-eosin and immunohistochemistry stainings. Compared with the clopidogrel group, the number of total inflammatory cells per mm2 thrombus area in the ticagrelor group was decreased by 28% (P = 0.009). The numbers of neutrophils and myeloperoxidase-positive cells per mm2 thrombus area in the ticagrelor group were respectively decreased by 35% (P = 0.016) and 28% (P = 0.047), as compared with those in the clopidogrel group. Moreover, ticagrelor treatment reduced the ratio of monocytes number higher than 250 per mm2 thrombus area compared with clopidogrel treatment (4% versus 29%, P = 0.048). In patients with undergoing PCI for STEMI, the loading dose ticagrelor regimen was associated with a reduction in inflammatory cell infiltration within thrombus compared with the loading dose clopidogrel regimen.

Myeloperoxydase and CD15 With Glycophorin C Double Staining in the Evaluation of Skin Wound Vitality in Forensic Practice.

BackgroundThe determination of skin wound vitality based on tissue sections is a challenge for the forensic pathologist. Histology is still the gold standard, despite its low sensitivity. Immunohistochemistry could allow to obtain a higher sensitivity. Upon the candidate markers, CD15 and myeloperoxidase (MPO) may allow to early detect polymorphonuclear neutrophils (PMN). The aim of this study was to evaluate the sensitivity and the specificity of CD15 and MPO, with glycophorin C co-staining, compared to standard histology, in a series of medicolegal autopsies, and in a human model of recent wounds.MethodsTwenty-four deceased individuals with at least one recent open skin wound were included. For each corpse, a post-mortem wound was performed in an uninjured skin area. At autopsy, a skin sample from the margins of each wound and skin controls were collected (n = 72). Additionally, the cutaneous surgical margins of abdominoplasty specimens were sampled as a model of early intravital stab wound injury (scalpel blade), associated with post-devascularization wounds (n = 39). MPO/glycophorin C and CD15/glycophorin C immunohistochemical double staining was performed. The number of MPO and CD15 positive cells per 10 high power fields (HPF) was evaluated, excluding glycophorin C—positive areas.ResultsWith a threshold of at least 4 PMN/10 high power fields, the sensitivity and specificity of the PMN count for the diagnostic of vitality were 16 and 100%, respectively. With MPO/glycophorin C as well as CD15/glycophorin C IHC, the number of positive cells was significantly higher in vital than in non-vital wounds (p < 0.001). With a threshold of at least 4 positive cells/10 HPF, the sensitivity and specificity of CD15 immunohistochemistry were 53 and 100%, respectively; with the same threshold, MPO sensitivity and specificity were 28 and 95%.ConclusionWe showed that combined MPO or CD15/glycophorin C double staining is an interesting and original method to detect early vital reaction. CD15 allowed to obtain a higher, albeit still limited, sensitivity, with a high specificity. Confirmation studies in independent and larger cohorts are still needed to confirm its accuracy in forensic pathology.

Milk Fat Globule Membrane Alleviates Short Bowel Syndrome-Associated Liver Injury in Rats Through Inhibiting Autophagy and NLRP3 Inflammasome Activation.

BackgroundThe milk fat globule membrane (MFGM), a tri-layer membrane structure surrounding the milk fat globule, has been shown to have immune-modulating properties. This study aimed to investigate the effects of MFGM supplementation in a rat model of short bowel syndrome (SBS) associated liver disease and its possible mechanisms.Materials and MethodsTwenty one male Sprague-Dawley rats were randomly divided into three groups: Sham, SBS (underwent massive small bowel resection), and SBS+MFGM (SBS rats supplemented with 1.5 g/kg/d MFGM). Liver pathology, myeloperoxidase (MPO) staining, serum levels of aspartate aminotransferase (AST)/alanine aminotransferase (ALT), endotoxin concentration, protein expression of autophagy and nucleotide binding oligomerization domain, leucine-rich repeat and pyrin domain-containing protein 3 (NLRP3) pathway in the liver tissue were measured.ResultsBoth SBS and SBS + MFGM groups had higher serum levels of ALT and liver endotoxin levels than the Sham group (P < 0.05), with no difference detected between each other. Compared with the SBS group, the SBS+MFGM group showed lower liver pathology scores of steatosis and inflammation, less MPO positive cells and reduced expressions of NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), Caspase-1, interleukin (IL)-1β(P < 0.05) in the liver. Additionally, the expression of Beclin-1 and microtubule-associated protein1 light chain 3(LC3) B, the fluorescence intensity of NLRP3 and LC3B in the SBS + MFGM group were lower than the SBS group (P < 0.05). The LC3B expression was positively correlated with the NLRP3 level.ConclusionEnteral supplementation of MFGM help to alleviate liver injury in SBS rats, which might be related to inhibition of aberrant activation of autophagy and NLRP3 inflammasome pathways.

Hepatocyte-specific deletion of adipose triglyceride lipase (adipose triglyceride lipase/patatin-like phospholipase domain containing 2) ameliorates dietary induced steatohepatitis in mice.

Increased fatty acid (FA) flux from adipose tissue to the liver contributes to the development of NAFLD. Because free FAs are key lipotoxic triggers accelerating disease progression, inhibiting adipose triglyceride lipase (ATGL)/patatin-like phospholipase domain containing 2 (PNPLA2), the main enzyme driving lipolysis, may attenuate steatohepatitis. Hepatocyte-specific ATGL knockout (ATGL LKO) mice were challenged with methionine-choline-deficient (MCD) or high-fat high-carbohydrate (HFHC) diet. Serum biochemistry, hepatic lipid content and liver histology were assessed. Mechanistically, hepatic gene and protein expression of lipid metabolism, inflammation, fibrosis, apoptosis, and endoplasmic reticulum (ER) stress markers were investigated. DNA binding activity for peroxisome proliferator-activated receptor (PPAR) α and PPARδ was measured. After short hairpin RNA-mediated ATGL knockdown, HepG2 cells were treated with lipopolysaccharide (LPS) or oleic acid:palmitic acid 2:1 (OP21) to explore the direct role of ATGL in inflammation in vitro. On MCD and HFHC challenge, ATGL LKO mice showed reduced PPARα and increased PPARδ DNA binding activity when compared with challenged wild-type (WT) mice. Despite histologically and biochemically pronounced hepatic steatosis, dietary-challenged ATGL LKO mice showed lower hepatic inflammation, reflected by the reduced number of Galectin3/MAC-2 and myeloperoxidase-positive cells and low mRNA expression levels of inflammatory markers (such as IL-1β and F4/80) when compared with WT mice. In line with this, protein levels of the ER stress markers protein kinase R-like endoplasmic reticulum kinase and inositol-requiring enzyme 1α were reduced in ATGL LKO mice fed with MCD diet. Accordingly, pretreatment of LPS-treated HepG2 cells with the PPARδ agonist GW0742 suppressed mRNA expression of inflammatory markers. Additionally, ATGL knockdown in HepG2 cells attenuated LPS/OP21-induced expression of proinflammatory cytokines and chemokines such as chemokine (C-X-C motif) ligand 5, chemokine (C-C motif) ligand (Ccl) 2, and Ccl5. Low hepatic lipolysis and increased PPARδ activity in ATGL/PNPLA2 deficiency may counteract hepatic inflammation and ER stress despite increased steatosis. Therefore, lowering hepatocyte lipolysis through ATGL inhibition represents a promising therapeutic strategy for the treatment of steatohepatitis.

RNA sequencing reveals induction of specific renal inflammatory pathways in a rat model of malignant hypertension

In malignant hypertension, far more severe kidney injury occurs than in the “benign” form of the disease. The role of high blood pressure and the renin–angiotensin–aldosterone system is well recognized, but the pathogenesis of the renal injury of malignant hypertension (MH) remains incompletely understood. Using the rat model of two-kidney, one-clip renovascular hypertension in which some but not all animals develop MH, we performed a transcriptomic analysis of gene expression by RNA sequencing to identify transcriptional changes in the kidney cortex specific for MH. Differential gene expression was assessed in three groups: MH, non-malignant hypertension (NMH), and normotensive, sham-operated controls. To distinguish MH from NMH, we considered two factors: weight loss and typical renovascular lesions. Mean blood pressure measured intraarterially was elevated in MH (220 ± 6.5 mmHg) as well as in NMH (192 ± 6.4 mmHg), compared to controls (119 ± 1.7 mmHg, p < 0.05). Eight hundred eighty-six genes were exclusively regulated in MH only. Principal component analysis revealed a separated clustering of the three groups. The data pointed to an upregulation of many inflammatory mechanisms in MH including pathways which previously attracted relatively little attention in the setting of hypertensive kidney injury: Transcripts from all three complement activation pathways were upregulated in MH compared to NMH but not in NMH compared with controls; immunohistochemistry confirmed complement deposition in MH exclusively. The expression of chemokines attracting neutrophil granulocytes (CXCL6) and infiltration of myeloperoxidase-positive cells were increased only in MH rats. The data suggest that these pathways, especially complement deposition, may contribute to kidney injury under MH.Key messagesThe most severe hypertension-induced kidney injury occurs in malignant hypertension.In a rat model of malignant hypertension, we assessed transcriptional responses in the kidney exposed to high blood pressure. A broad stimulation of inflammatory mechanisms was observed, but a few specific pathways were activated only in the malignant form of the disease, notably activation of the complement cascades.Complement inhibitors may alleviate the thrombotic microangiopathy of malignant hypertension even in the absence of primary complement abnormalities.

Sex-dependent effects of genetic upregulation of activated protein C on delayed effects of acute radiation exposure in the mouse heart, small intestine, and skin.

Accidental exposure to ionizing radiation may lead to delayed effects of acute radiation exposure (DEARE) in many organ systems. Activated protein C (APC) is a known mitigator of the acute radiation syndrome. To examine the role of APC in DEARE, we used a transgenic mouse model with 2- to 3-fold increased plasma levels of APC (high in APC, APCHi). Male and female APCHi mice and wild-type littermates were exposed to 9.5 Gy γ-rays with their hind-legs (bone marrow) shielded from radiation to allow long-term survival. At 3 and 6 months after irradiation, cardiac function was measured with ultrasonography. At 3 months, radiation increased cardiac dimensions in APCHi males, while decreases were seen in wild-type females. At this early time point, APCHi mice of both sexes were more susceptible to radiation-induced changes in systolic function compared to wild-types. At 6 months, a decrease in systolic function was mainly seen in male mice of both genotypes. At 6 months, specimens of heart, small intestine and dorsal skin were collected for tissue analysis. Female APCHi mice showed the most severe radiation-induced deposition of cardiac collagens but were protected against a radiation-induced loss of microvascular density. Both male and female APCHi mice were protected against a radiation induced upregulation of toll-like receptor 4 in the heart, but this did not translate into a clear protection against immune cell infiltration. In the small intestine, the APCHi genotype had no effect on an increase in the number of myeloperoxidase positive cells (seen mostly in females) or an increase in the expression of T-cell marker CD2 (males). Lastly, both male and female APCHi mice were protected against radiation-induced epidermal thickening and increase in 3-nitrotyrosine positive keratinocytes. In conclusion, prolonged high levels of APC in a transgenic mouse model had little effects on indicators of DEARE in the heart, small intestine and skin, with some differential effects in male compared to female mice.

Induction of CCN1 in Growing Saccular Aneurysms: A Potential Marker Predicting Unstable Lesions.

Growing evidence has suggested that inflammatory responses promote the progression of saccular intracranial aneurysms (IAs). However, a biomarker predicting the progression has yet to be established. This study aimed to identify novel molecules upregulated during the progression using a previously established rat aneurysm model. In this model, aneurysms are induced at the surgically created common carotid artery (CCA) bifurcation. Based on sequential morphological data, the observation periods after the surgical manipulations were defined as the growing phase (on the 10th day) or the stable phase (on the 30th day). Total cell lysates from the CCA with or without an aneurysm lesion were prepared to perform protein array analysis. The protein array analysis revealed that the matricellular protein cellular communication network factor 1 (CCN1) is induced in lesions during the growing phase. Immunohistochemistry corroborated the significant upregulation of CCN1 in the growing phase compared with the stable phase. Simultaneously with the induction of CCN1, significant increases in the number of CD68-positive macrophages, myeloperoxidase-positive cells, and proliferating smooth muscle cells in lesions were observed. Immunohistochemistry of human IA specimens reproduced the induction of CCN1 in some lesions. These findings imply a potential role of CCN1 as a marker predicting the progression of saccular aneurysms.

Sex-dependent mechanisms involved in renal tolerance to ischemia-reperfusion: Role of inflammation and histone H3 citrullination

Ischemia-reperfusion (I/R) injury, an inevitable result of kidney transplantation, triggers early inflammatory events that affect graft viability. Evidence from human transplantation and preclinical models of I/R suggests that a female hormonal environment positively influences the ability to recover from ischemic injury. However, the mechanisms behind these effects remain mostly unexplored. Here, we studied the influence of sex on pro-inflammatory mediators involved in the pathophysiology of acute I/R injury in male, female, and female ovariectomized (OVX) Wistar rats that underwent unilateral renal ischemia for 45 min, followed by 24 h of reperfusion. We found improved renal function, reduced cytokine expression, and decreased infiltration of myeloperoxidase-positive cells in females after I/R, when compared to their male and female OVX counterparts. Remarkably, citrullination of histone H3 was exacerbated in serum and renal tubules of females after I/R. In contrast, we observed lower levels of citrullinated histone H3 in male and female OVX rats in response to I/R, mostly in neutrophil extracellular traps. Our results demonstrate that female sex promotes renal I/R tolerance by attenuating pro-inflammatory mediators involved in I/R-induced damage.

Synbiotics administration leads to attenuated mucosal inflammatory neutrophil infiltration and increased hematocrit in experimental ulcerative colitis.

The objective of the study was to estimate the effects of synbiotics on laboratory, macroscopic, and histopathologic features in dextran sulfate sodium (DSS) experimental colitis. A total of 40 Wistar rats received 5% of DSS in their drinking water for 8 days to induce ulcerative colitis (UC). Eight rats were sacrificed to confirm the presence of UC. The remaining rats were randomly assigned to two groups: the synbiotics group, which received synbiotics once per day and the control group, which received tap water for another 8 days. On the 8th day of DSS administration animals developed UC with bloody diarrhea. In the majority of the hematologic variables studied (hemoglobin [HB], red blood cells, platelets, mean corpuscular volume, and mean corpuscular HB), in bodyweight and histopathologic colitis score there was no significant difference between groups. However, the synbiotics group, compared to control, presented a significantly greater colon length on the 4th day, significantly increased hematocrit (HT) on the 8th day, and a significantly decreased number of myeloperoxidase positive cells on the 8th day. Furthermore, there was a trend toward histopathological and clinical improvement. Administration of synbiotics in the experimental UC results in an attenuation of mucosal inflammatory neutrophil infiltration and an increase in HT.

MUC5B induces in vitro neutrophil extracellular trap formation: Implication in otitis media.

BackgroundChronic otitis media (COM) is characterized by middle ear fluid predominantly containing cytokines, Nontypeable haemophilus influenzae (NTHi), the mucin MUC5B, and neutrophil extracellular traps (NETs). NETs consist of extracellular DNA coated with antibacterial proteins such as myeloperoxidase (MPO) and citrullinated histone 3 (CitH3). NETs can damage tissues and sustain inflammation. Our study aimed to develop an in vitro model of NETosis, testing COM inductors.MethodsNETosis was evaluated in fresh blood human neutrophils attached to collagen‐coated plates and in suspension exposed to phorbol myristate acetate (PMA) as a control, and COM relevant mediators. Confocal microscopy, DNA fluorescence assay and flow cytometry were used to quantify NETosis.ResultsPMA exposure induced DNA, MPO, and CitH3 by immunofluorescence (IF) most significantly at 3 hours (3.8‐fold for DAPI, 7.6‐fold for MPO, and 6.9‐fold for CitH3, all P < .05). IL‐8 and TNF‐α cytokines showed milder increases of DAPI, MPO, and CitH3 positive cells. NTHi had no effect on these NETs markers. Purified salivary MUC5B (10 to 40 μg/mL) produced potent increases, comparable to PMA. A composite NET score summing the fold‐increases for DAPI, MPO, and CitH3 demonstrated PMA at 13.6 to 19 relative to control set at 1; and MUC5B at 8.6 to 16.3 (all P < .05). IL‐8 and TNF‐α showed scores of 5.4 and 3, respectively, but these were not statistically significant.ConclusionWe developed a reliable in vitro assay for NETosis which demonstrated that salivary MUC5B is a potent inductor of NETs whereas IL‐8, TNF‐α, live and lyzed NTHi demonstrated minimal to no NETosis.Level of evidenceNA.