A microfluidic device for assessment of E-selectin-mediated neutrophil recruitment to inflamed endothelium and prediction of therapeutic response in sickle cell disease.

Neutrophil recruitment to the inflamed endothelium is a multistep process and is of utmost importance in the development of the hallmark vaso-occlusive crisis in sickle cell disease (SCD). However, there lacks a standardized, clinically feasible approach for assessing neutrophil recruitment to the inflamed endothelium for individualized risk stratification and therapeutic response prediction in SCD. Here, we describe a microfluidic device functionalized with E-selectin, a critical endothelial receptor for the neutrophil recruitment process, as a strategy to assess neutrophil binding under physiologic flow in normoxia and clinically relevant hypoxia in SCD. We show that hypoxia significantly enhances neutrophil binding to E-selectin and promotes the formation of neutrophil-platelet aggregates. Moreover, we identified two distinct patient populations: a more severe clinical phenotype with elevated lactate dehydrogenase levels and absolute reticulocyte counts but lowered fetal hemoglobin levels associated with constitutively less neutrophil binding to E-selectin. Mechanistically, we demonstrate that the extent of neutrophil activation correlates with membrane L-selectin shedding, resulting in the loss of ligand interaction sites with E-selectin. We also show that inhibition of E-selectin significantly reduces leukocyte recruitment to activated endothelial cells. Our findings add mechanistic insight into neutrophil-endothelial interactions under hypoxia and provide a clinically feasible means for assessing neutrophil binding to E-selectin using clinical whole blood samples, which can help guide therapeutic decisions for SCD patients.

Comparative Assessment of Leukocyte Infiltrate Composition in the Uterine and Vaginal Tissues in Rats with Experimental Endomyometritis Treated with Interferon-γ at Different Times of the Day.

The cell composition of leukocyte infiltrates in the endometrium, myometrium, and vaginal walls was studied in Wistar rats with modeled chronic endomyometritis after administration of IFNγ (0.1 µg/100 g body weight) in different daily regimens (10.00 or 20.00). Morning injections of this cytokine ameliorated inflammatory infiltration of the uterine wall and vagina, but increased the content of neutrophils in the endometrium. Evening cytokine injections reduced neutrophilic infiltration, enhanced mononuclear infiltration, and had no effect on plasmacytic infiltration of the uterine and vaginal walls. In the vaginal wall, both IFNγ administration schedules decreased neutrophil content. The data indicate the necessity to take into account the circadian rhythms in IFN therapy.

Target-Specific Fluorescence-Mediated Tomography for Non-Invasive and Dynamic Assessment of Early Neutrophil Infiltration in Murine Experimental Colitis.

The role of neutrophils in the pathogenesis of inflammatory bowel disease (IBD) is still only incompletely understood. Here, we evaluated target-specific fluorescence-mediated tomography (FMT) for visualization of neutrophil infiltration in murine experimental DSS-induced colitis. Colitis was assessed using clinical, endoscopic, and histopathological parameters. Intestinal neutrophil infiltration was determined at day 0, 4, and 10 by targeted FMT after injection of a neutrophil-specific fluorescence-labelled monoclonal antibody (Gr-1). Complementary, immunofluorescence tissue sections with Gr-1 and ELISA-based assessment of tissue myeloperoxidase (MPO) served as the gold standard for the quantification of neutrophil infiltration. Colitic animals showed decreasing body weight, presence of fecal occult blood, and endoscopic signs of inflammation. FMT revealed a significantly increased level of fluorescence only four days after colitis induction as compared to pre-experimental conditions (pmol tracer 73.2 ± 18.1 versus 738.6 ± 80.7; p < 0.05), while neither body weight nor endoscopic assessment showed significant changes at this early time. Confirmatory, post-mortem immunofluorescence studies and measurements of tissue MPO confirmed the presence of increased neutrophil infiltration in colitic mice compared to controls. Concluding, Gr-1 targeted FMT can detect early colonic infiltration of neutrophils in experimental colitis even before clinical symptoms or endoscopic alterations occur. Therefore, FMT might be an important tool for repetitive and non-invasive monitoring of inflammatory cell infiltrate in intestinal inflammation.

Apoptosis signal-regulating kinase-1 promotes inflammasome priming in macrophages.

We previously showed that mice deficient in apoptosis signal-regulating kinase-1 (ASK1) were partially protected against ventilator-induced lung injury. Because ASK1 can promote both cell death and inflammation, we hypothesized that ASK1 activation regulates inflammasome-mediated inflammation. Mice deficient in ASK1 expression (ASK1-/-) exhibited significantly less inflammation and lung injury (as measured by neutrophil infiltration, IL-6, and IL-1ß) in response to treatment with inhaled lipopolysaccharide (LPS) compared with wild-type (WT) mice. To determine whether this proinflammatory response was mediated by ASK1, we investigated inflammasome-mediated responses to LPS in primary macrophages and bone marrow-derived macrophages (BMDMs) from WT and ASK1-/- mice, as well as the mouse alveolar macrophage cell line MH-S. Cells were treated with LPS alone for priming or LPS followed by ATP for activation. When macrophages were stimulated with LPS followed by ATP to activate the inflammasome, we found a significant increase in secreted IL-1ß from WT cells compared with ASK1-deficient cells. LPS priming stimulated an increase in NOD-like receptor 3 (NLRP3) and pro-IL-1ß in WT BMDMs, but expression of NLRP3 was significantly decreased in ASK1-/- BMDMs. Subsequent ATP treatment stimulated an increase in cleaved caspase-1 and IL-1ß in WT BMDMs compared with ASK1-/- BMDMs. Similarly, treatment of MH-S cells with LPS + ATP caused an increase in both cleaved caspase-1 and IL-1ß that was diminished by the ASK-1 inhibitor NQDI1. These results demonstrate, for the first time, that ASK1 promotes inflammasome priming.

Assessment of mucosal integrity by quantifying neutrophil granulocyte influx in murine models of acute intestinal injury.

Intact epithelial body surfaces represent physical barriers which protect the organism from invading pathogens and loss of nutrients. Barrier malfunction is closely linked to disorders such as inflammatory bowel disease and graft-versus-host disease. In fact, several pharmacological or radiobiological therapeutic strategies have side effects that affect epithelial surfaces. In this context, assays that accurately assess epithelial barrier integrity in patients and animal models are crucial to create a better understanding of the mechanisms leading to disease or limiting therapeutic approaches due to barrier disruption. Here, we tested the ability of the widely used FITC-dextran intestinal permeability analysis to evaluate loss of intestinal barrier integrity in different murine models of gut mucosal damage and established influx of neutrophil granulocytes into the intestinal lamina propria (LP) as an alternative approach. We demonstrate that the sensitivity and specificity of FITC-dextran intestinal permeability analysis is relatively low: Although it did represent severe forms of mucosal damage due to intensive conditioning therapy (high doses of either total body irradiation (TBI) or chemotherapy) or after conditioning and allogeneic stem cell transplantation, it did not recognize less severe forms of damage as after lower doses of TBI or chemotherapy alone. In addition, discrimination of untreated from irradiated mice by differences in FITC-dextran translocation was not exact. In contrast, influx of neutrophil granulocytes into the intestinal LP, which reflects immune activation due to translocation of microbes and microbial products during intestinal barrier breech, quantitatively correlated with the severity of intestinal barrier damage. It accurately represented both severe and less severe forms of intestinal damage as after high or lower dose TBI or chemotherapy and correctly discriminated treated from untreated animals. Taken together, we demonstrate the limitations of FITC-dextran intestinal permeability analysis and identify intestinal neutrophil influx as a powerful additional tool to measure breakdown of intestinal barrier function.

Characterization of a vraG Mutant in a Genetically Stable Staphylococcus aureus Small-Colony Variant and Preliminary Assessment for Use as a Live-Attenuated Vaccine against Intrammamary Infections.

Staphylococcus aureus is a leading cause of bovine intramammary infections (IMIs) that can evolve into difficult-to-treat chronic mastitis. To date, no vaccine formulation has shown high protective efficacy against S. aureus IMI, partly because this bacterium can efficiently evade the immune system. For instance, S. aureus small colony variants (SCVs) have intracellular abilities and can persist without producing invasive infections. As a first step towards the development of a live vaccine, this study describes the elaboration of a novel attenuated mutant of S. aureus taking advantage of the SCV phenotype. A genetically stable SCV was created through the deletion of the hemB gene, impairing its ability to adapt and revert to the invasive phenotype. Further attenuation was obtained through inactivation of gene vraG (SACOL0720) which we previously showed to be important for full virulence during bovine IMIs. After infection of bovine mammary epithelial cells (MAC-T), the double mutant (ΔvraGΔhemB) was less internalized and caused less cell destruction than that seen with ΔhemB and ΔvraG, respectively. In a murine IMI model, the ΔvraGΔhemB mutant was strongly attenuated, with a reduction of viable counts of up to 5-log10 CFU/g of mammary gland when compared to the parental strain. A complete clearance of ΔvraGΔhemB from glands was observed whereas mortality rapidly (48h) occurred with the wild-type strain. Immunization of mice using subcutaneous injections of live ΔvraGΔhemB raised a strong immune response as judged by the high total IgG titers measured against bacterial cell extracts and by the high IgG2a/IgG1 ratio observed against the IsdH protein. Also, ΔvraGΔhemB had sufficient common features with bovine mastitis strains so that the antibody response also strongly recognized strains from a variety of mastitis associated spa types. This double mutant could serve as a live-attenuated component in vaccines to improve cell-mediated immune responses against S. aureus IMIs.

Magnetic resonance imaging provides sensitive in vivo assessment of experimental ventilator-induced lung injury.

Animal models play a critical role in the study of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury (VILI). One limitation has been the lack of a suitable method for serial assessment of acute lung injury (ALI) in vivo. In this study, we demonstrate the sensitivity of magnetic resonance imaging (MRI) to assess ALI in real time in rat models of VILI. Sprague-Dawley rats were untreated or treated with intratracheal lipopolysaccharide or PBS. After 48 h, animals were mechanically ventilated for up to 15 h to induce VILI. Free induction decay (FID)-projection images were made hourly. Image data were collected continuously for 30 min and divided into 13 phases of the ventilatory cycle to make cinematic images. Interleaved measurements of respiratory mechanics were performed using a flexiVent ventilator. The degree of lung infiltration was quantified in serial images throughout the progression or resolution of VILI. MRI detected VILI significantly earlier (3.8 ± 1.6 h) than it was detected by altered lung mechanics (9.5 ± 3.9 h, P = 0.0156). Animals with VILI had a significant increase in the Index of Infiltration (P = 0.0027), and early regional lung infiltrates detected by MRI correlated with edema and inflammatory lung injury on histopathology. We were also able to visualize and quantify regression of VILI in real time upon institution of protective mechanical ventilation. Magnetic resonance lung imaging can be utilized to investigate mechanisms underlying the development and propagation of ALI, and to test the therapeutic effects of new treatments and ventilator strategies on the resolution of ALI.

Cytological assessment of the epithelial cells of the nasal mucous membrane after local fluticasone therapy.

The majority of cytological studies concern the influence of glucocorticosteroids on cells involved in creating and sustaining inflammation, such as eosinophils or neutrophils. Much less attention is devoted to epithelial cells. It should also be noticed that glucocorticosteroid drugs administered nasally for local action can significantly change the cytological image of the nasal mucous membrane. Therefore, the purpose of this research was to cytologically assess the influence of topical fluticasone therapy on the nasal mucous membrane cells, with special attention for the changes in the morphology of epithelial cells. The research samples were taken from patients with symptoms of chronic rhinitis and suspected allergies. The research was a two-step process. In the first step, a smear was taken from the surface of the nasal mucous membrane of the above-mentioned patients before the start of therapy and the obtained cytological image was compared with a control image of the nasal mucous of healthy people. Step two involved the cytology of the same patients after 4 weeks of fluticasone therapy, applied as a nasal aerosol in two doses of 50 µg to each nostril once per day, in the combined daily dose of 200 µg (for adults and children aged 12 or more). Children aged between 4 and 12 were given a single dose of 50 µg to each nostril once per day, in a daily dose of 100 µg. Based on smears stained according to the Papanicolaou and Pappenheim method, a qualitative and quantitative analysis of changes in the mucous membrane of nasal cells was performed. The cytological assessment of nasal mucous membrane stains of patients with chronic rhinitis before fluticasone treatment enabled a diagnosis of chronic infectious rhinitis, compared through the presence of numerous neutrophils and bacteria. The studied samples did not show significant changes in the morphology of epithelial cells, only a few cells with mild vacuolation changes of the cytoplasm were found. The use of fluticasone, however, caused a significant decrease in the neutrophilia and the appearance of numerous epithelial cells with intensified cytoplasm vacuolation in the sample. The results obtained allow us to conclude that standard fluticasone therapy as administered nasally in aerosol form to patients with diagnosed nonallergic nasal mucous membrane inflammation caused a significant reduction in the inflammation without showing cytological characteristics of damage to the epithelium of the nasal mucous membrane. The intensified vacuolation observed in the cytoplasm of epithelial cells, most prominently in the columnar cells, might suggest the stimulation of autophagic processes.

In Vitro Assessment of Human Islet Vulnerability to Instant Blood-Mediated Inflammatory Reaction (IBMIR) and Its Use to Demonstrate a Beneficial Effect of Tissue Culture.

Culture of human pancreatic islets is now routinely carried out prior to clinical islet allotransplantation, using conditions that have been developed empirically. One of the major causes of early islet destruction after transplantation is the process termed instant blood-mediated inflammatory reaction (IBMIR). The aim of this study was to develop in vitro methods to investigate IBMIR and apply them to the culture conditions used routinely in our human islet isolation laboratory. Freshly isolated or precultured (24 h, 48 h) human islets were incubated in either ABO-compatible allogeneic human blood or Hank's buffered salt solution (HBSS) for 1 h at 37°C. Tissue factor (TF) expression and leukocyte migration were assessed by light microscopy. TF was also quantified by ELISA. To assess ß-cell function, glucose-stimulated insulin secretion (GSIS) assay was carried out. The extent of islet ß-cell damage was quantified using a proinsulin assay. Islets cultured for 24 h had higher GSIS when compared to freshly isolated or 48-h precultured islets. Freshly isolated islets had significantly higher TF content than 24-h and 48-h precultured islets. Incubation of freshly isolated human islets in allogeneic human blood released 6.5-fold higher level of proinsulin in comparison to freshly isolated human islets in HBSS. The high level of proinsulin released was significantly attenuated when precultured islets (24 h or 48 h) were exposed to fresh blood. Histological examination of fresh islets in blood clot showed that some islets were fragmented, showing signs of extraislet insulin leakage and extensive neutrophil infiltration and necrosis. These features were markedly reduced when the islets were cultured for 24 h. These results suggest that our standard 24-h islet culture is markedly beneficial in attenuating IBMIR, as evidenced by increased GSIS, lower content of TF, decrease islet fragmentation, and proinsulin release.

Assessment of pulmonary toxicity of MgO nanoparticles in rats.

In this study, we have evaluated the pulmonary toxicity of MgO nanoparticles (MgO NPs) in rats following their exposure. NPs in phosphate buffered saline + 1% Tween 80 were exposed via intratracheal instillation at a doses of 1 mg/kg or 5 mg/kg into rat lungs and evaluated for various tissue damage markers like alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) in bronchoalveolar lavage (BAL) fluid and histopathology of lungs at 1, 7, and 30 days of post-exposure intervals. A dose-dependant increase in ALP and LDH activity was observed in BAL fluids of rat lungs than sham control at all post-exposure periods (P <0.05), and a dose-dependant infiltration of interstitial lymphocytes, peribronchiolar lymphocytic infiltration, and dilated and/or congested vessels at 1 day post-exposure period, worsened at 1 week period, and were reduced at 1 month at histology, indicating the pulmonary toxicity of MgO NPs. In conclusion, MgO NPs exposure produced a dose-dependent pulmonary toxicity in rats and was comparable with that of Quartz particles.

Longitudinal assessment of imatinib's effect on the blood-brain barrier after ischemia/reperfusion injury with permeability MRI.

Acute ischemic stroke (AIS) often results in degeneration of the blood-brain barrier (BBB), which can lead to vasogenic edema and an increased risk of intracerebral hemorrhage. Imatinib is an agent that may be able to protect the BBB and reduce the risk of the harmful consequences of BBB degeneration. We sought to measure the effect of Imatinib on the BBB after experimental stroke longitudinally in vivo with permeability dynamic contrast-enhanced MRI. Ischemia/reperfusion injury was induced with a transient middle cerebral artery occlusion surgery. Rats were given Imatinib at 2 and 20 h after stroke onset. Post-assessment included neurologic functioning, MR imaging, Evans Blue extravasation, Western blot, and immunohistology assay. Imatinib protected the BBB by 24 h but failed to decrease BBB permeability at an earlier time-point. Imatinib also reduced infarct volume, edema, and improved neurologic functioning by 24 h. Rats treated with Imatinib also had a higher expression of the BBB structural protein Zona ocludens-1 and a reduction in nuclear factor-kappa beta (NF-κß) activation. Imatinib is a promising agent to protect the BBB after AIS, but its effect on the BBB may not become prominent until 24 h after the onset of ischemia. This finding may help elucidate Imatinib's role in the clinical management of AIS and influence future study designs.

Toxicity assessment of air-delivered particle-bound polybrominated diphenyl ethers.

Human exposure to polybrominated diphenyl ethers (PBDEs) can occur via ingestion of indoor dust, inhalation of PBDE-contaminated air and dust-bound PBDEs. However, few studies have examined the pulmonary toxicity of particle-bound PBDEs, mainly due to the lack of an appropriate particle-cell exposure system. In this study we developed an in vitro exposure system capable of generating particle-bound PBDEs mimicking dusts containing PBDE congeners (BDEs 35, 47 and 99) and delivering them directly onto lung cells grown at an air-liquid interface (ALI). The silica particles and particles-coated with PBDEs ranged in diameter from 4.3 to 4.5 µm and were delivered to cells with no apparent aggregation. This experimental set up demonstrated high reproducibility and sensitivity for dosing control and distribution of particles. ALI exposure of cells to PBDE-bound particles significantly decreased cell viability and induced reactive oxygen species generation in A549 and NCI-H358 cells. In male Sprague-Dawley rats exposed via intratracheal insufflation (0.6 mg/rat), particle-bound PBDE exposures induced inflammatory responses with increased recruitment of neutrophils to the lungs compared to sham-exposed rats. The present study clearly indicates the potential of our exposure system for studying the toxicity of particle-bound compounds.

Assessment of the relationship between Helicobacter pylori infection, endoscopic appearance and histological changes of the gastric mucosa in children with gastritis (a single center experience).

BACKGROUND: Helicobacter pylori infection is an important cause of gastritis in childhood, its role in the pathogenesis of peptic ulcer disease in adults and children being generally known. In some cases, there are therapeutic management issues, because they do not heal or they often relapse, although treatment regimens are applied as recommended. Our aim was to analyze the relationship between endoscopic appearance and histological changes of the gastric mucosa in children with gastritis associated with H. pylori infection, in which persistent infection after treatment was found. MATERIALS AND METHODS: It was a prospective study on 1332 children assessed in our Service (Ist Pediatric Clinic, Tirgu Mures, Romania), between January 2008 and January 2013, for gastritis with various etiologies. There were 609 cases of gastritis-associated with H. pylori infection. RESULTS: The average age of patients was 13.21 years; the higher incidence was noted in 13-18-year-old group, female gender and rural areas provenience; a number of 544 patients diagnosed with gastritis with H. pylori were reassessed subsequently; after treatment, gastritis has healed and the infection was eradicated in 88.23% cases after a month, while in 64 patients infection persisted. After a second regimen, endoscopic-histological modifications persisted in 31 (5.69%) cases; 1.28% cases remained positive for longer. CONCLUSIONS: H. pylori infection was associated with high age group, as well as with endoscopic modifications; also, the presence of H. pylori was correlated with histopathologic diagnostic. We try to emphasize the importance of assessing bacterial resistance to antibiotics, studying of bacterial genome and genetic susceptibility of human subjects.

Sample size determination in two-sided distribution-free treatment versus control multiple comparisons.

The problem of power and sample size determination for distribution-free multiple comparison tests of K treatments versus a control group is addressed. We define the power as the probability of correctly rejecting one specified or all K hypotheses, corresponding to the per-pair and all-pairs power, respectively. The power formulas are derived for both joint ranking and pairwise ranking mechanism for general multiple comparison problems, followed by explicit form of these formulas when the single-step, step-down, or step-up adjustments are applied. The proposed power and sample size calculation methods apply to scenarios both when the underlying distributions are known and when they are unknown but a pilot study is available. Numerical methods via quasi-Monte Carlo integration and Monte Carlo integration are assessed. Our simulation studies show the accuracy of the power and sample size calculation formulas. We recommend the Monte Carlo integration as the calculation algorithm. An example from a mouse peritoneal cavity study is used to demonstrate the application of the methods.

Recent advances in autoimmune pancreatitis: type 1 and type 2.

Autoimmune pancreatitis (AIP) is a form of chronic pancreatitis characterised clinically by frequent presentation with obstructive jaundice, histologically by a lymphoplasmacytic infiltrate with fibrosis, and therapeutically by a dramatic response to steroids. When so defined, AIP can be sub-classified into two subtypes, 1 and 2. Recent international consensus diagnostic criteria for AIP have been developed for diagnosis of both forms of AIP. Type 1 AIP is the pancreatic manifestation of a multiorgan disease, recently named IgG4-related disease. Little is known about the pathogenesis of either form of AIP. Despite frequent association of type 1 AIP with elevated serum IgG4 levels and infiltration with IgG4-positive plasma cells, it is unlikely that IgG4 plays a pathogenic role in AIP. Type 1 AIP responds to steroids, but there needs to be consensus on treatment regimens for induction and therapeutic end points. Relapses are common, but can be reduced by long-term use of low-dose steroids. Recent reports suggest that immunomodulators (azathioprine, 6-mercaptopurine and mycophenolate mofetil), as well biological agents (the antibody to CD20, rituximab) may have a role in maintaining remission in relapsing type 1 AIP. Future studies should clarify the best management options for treatment of relapses and maintenance of remission. Type 2 AIP is a pancreas-specific disorder not associated with IgG4. It presents in younger individuals equally with obstructive jaundice and pancreatitis. The inflammatory process responds to steroid therapy; relapses are uncommon. The clinical spectrum and long-term outcomes of medically treated type 2 AIP are still being evaluated.

Assessment of the respiratory sensitization potential of proteins using an enhanced mouse intranasal test (MINT).

There remains a need for a simple and predictive animal model to identify potential respiratory sensitizers. The mouse intranasal test (MINT) was developed to assess the relative allergic potential of detergent enzymes, however, the experimental endpoints were limited to evaluation of antibody levels. The present study was designed to evaluate additional endpoints (serum and allergic antibody levels, pulmonary inflammation and airway hyperresponsiveness (AHR)) to determine their value in improving the predictive accuracy of the MINT. BDF1 mice were intranasally instilled on days 1, 3, 10, 17 and 24 with subtilisin, ovalbumin, betalactoglobulin, mouse serum albumin or keyhole limpet hemocyanin; challenged with aerosolized methacholine or the sensitizing protein on day 29 to assess AHR, and sacrificed on day 29 or 30. Under the conditions of this study, evaluation of AHR did not improve the predictive power of this experimental model. Allergic antibody responses and IgG isotype characterization proved to be the most sensitive and reliable indicators of the protein allergenic potential with BAL responses providing additional insight. These data highlight that the evaluation of the respiratory sensitization potential of proteins can be best informed when multiple parameters are evaluated and that further improvements and refinements of the assay are necessary.

Assessment of gingival mucosa of infant rats during teething.

AIM: The aim of the present study was to perform a histological analysis of the gingival mucosa in infant rats undergoing the teething process. MATERIALS AND METHODS: Eighteen Wistar rats between 8 and 15 days of life were distributed among three groups: group A--without teething; group B--eruption of incisors; and group C--eruption of incisors and molars. The samples included teeth and periodontal tissue from the region of the incisors and molars of each animal. Fragments were processed for histological analysis and submitted to immunohistochemical analysis. RESULTS: In the 8-day-old rats, mild inflammatory infiltrate predominated with mononuclear cells in the pericoronal follicles of the incisors and molars. At 12 days of age, all animals exhibited moderate inflammation in the pericoronal follicles and epithelium of the incisors and mild inflammatory infiltrate with predominantly mononuclear cells in the molars. At 15 days of age, moderate neutrophilic exudate was found in the pericoronal follicles and epithelium of the incisors and molars. Immunohistochemical analysis revealed positivity for interleukin- 1b in the pericoronal follicles in the pre-eruption phase. CONCLUSION: An inflammatory reaction with progressive intensity occurs during the teething process, the response of which is preceded by the release of interleukin-1b. CLINICAL SIGNIFICANCE: Morphological proof of events that occur during teething that can affect the dynamics of the physiologic process manifesting as clinical symptoms.

Neutrophilic infiltration in gluten-sensitive enteropathy is neither uncommon nor insignificant: assessment of duodenal biopsies from 267 pediatric and adult patients.

The histologic findings of celiac disease, that is, gluten-sensitive enteropathy (GSE), are dominated by increased intraepithelial lymphocytes, villous blunting, lymphoplasmacytic infiltration of lamina propria, and crypt hyperplasia. To date, neutrophils have not been thought to constitute a significant cell type in GSE, and their presence often invokes consideration of alternative diagnoses. Thus, we sought to determine the prevalence and severity of neutrophilic infiltration in duodenal biopsies from patients with GSE. The degree of neutrophilic infiltration and features characteristic of GSE were assessed in duodenal biopsies from 267 clinically confirmed GSE patients (116 adults and 151 children). These specimens were graded by the disease activity score (DAS) and the neutrophilic activity score (NAS). Gastric antral biopsies obtained from 195 patients were also evaluated for lymphocytic gastritis. NAS was correlated with DAS and other clinicopathologic features. We found that 56% of pediatric and 28% of adult GSE patients had significant duodenal neutrophilia. NAS was higher in children than in adults (2.3 vs. 1.2, P<0.001). Multivariate regression showed that DAS, eosinophilic infiltration, and foveolar metaplasia correlated positively, and age correlated negatively with NAS. Lymphocytic gastritis was seen in 21.5% of the gastric biopsies. The presence of lymphocytic gastritis correlated positively with NAS and DAS, and in the pediatric population it correlated negatively with age. Significant duodenal neutrophilia is often found in patients with celiac disease, especially in the pediatric population, and is associated with more active disease. Thus, the findings of duodenal neutrophilia in biopsies, otherwise consistent with GSE, should not preclude the diagnosis of GSE.

In vivo assessment of macrophage CNS infiltration during disruption of the blood-brain barrier with focused ultrasound: a magnetic resonance imaging study.

Focused ultrasound has been discovered to locally and reversibly increase permeability of the blood-brain barrier (BBB). However, inappropriate sonication of the BBB may cause complications, such as hemorrhage and brain tissue damage. Tissue damage may be controlled by selecting optimal sonication parameters. In this study, we sought to investigate the feasibility of labeling cells with superparamagnetic iron oxide particles to assess the inflammatory response during focused-ultrasound-induced BBB opening. We show that infiltration of phagocytes does not occur using optimal parameters of sonication. Taken together, the results of our study support the usefulness and safety of focused-ultrasound-induced BBB opening for enhancing drug delivery to the brain. These findings may have implications for the optimization of sonication parameters.

Mold fabrication and biological assessment of porous DNA-chitosan complexes.

A previous study revealed that DNA-chitosan complex prepared from the reaction between native DNA and chitosan in aqueous solution has suitable porosity for cell seeding, is nontoxic, and causes only a mild soft-tissue response. This simple and easy fabrication method for porous DNA-chitosan complex provides for a wide variety of applications as a scaffold material. The present study evaluated whether rinsing with PBS solution can fabricate DNA-chitosan complex in a mold and the histopathological responses of rat soft tissues to fabricated DNA-chitosan complexes. DNA-chitosan complex paste was prepared by mixing distilled water and freeze-dried water-rinsed DNA-chitosan complex powder. A DNA-chitosan complex disk could be fabricated by rinsing with PBS buffer and subsequently freeze-drying the DNA-chitosan complex paste in the mold. Thus, a wide range of applications of DNA-chitosan complex for tissue engineering can be anticipated using the present easy fabrication method. The porosity of the disk was 85%, and many pores were visible in the DNA-chitosan complex (before fabrication) and in the fabricated DNA-chitosan disk. The values of the complex disks gradually reduced in the tissues although 60% of disks remained in the tissues. In conclusion, an easy fabrication method for making porous DNA-chitosan complex disks was developed. It was found that the fabrication method can delay the biodegradation of the DNA-chitosan complex disk without serious tissue responses in vivo. DNA-chitosan complex is promising as a scaffold material, and a wide range of applications of DNA-chitosan complex for tissue engineering are anticipated.