[Introduction of a decalcification method for bone marrow biopsy tissue].

Bone marrow biopsy is one of the important means of hematopathological diagnosis, which has decisive diagnostic significance for various benign and malignant lymphohematopoietic system diseases. Its diagnostic value includes morphological observation, immunohistochemistry, genetics, and molecular biology testing. Owing to the unique nature of bone marrow biopsy, decalcification is an essential step in the pre-treatment process. Its purpose is to remove calcium from bone tissue, preserve intact collagen fiber components, facilitate tissue sectioning, and prevent tissue detachment during staining. If bone marrow biopsy lacks sufficient decalcification, preparing a section is difficult. Conversely, if decalcification is excessive, it can seriously disrupt tissue antigen activity. Therefore, a decalcification method with high decalcification efficiency and mild antigen damage is essential for bone marrow biopsy. This article introduces a bone marrow biopsy tissue decalcification method with high efficiency and less antigen loss: decalcification is performed at room temperature with 12% formic acid and 8% hydrochloric acid decalcification solution on a shaker.

Case report: Autoimmune encephalomyelitis following cytomegalovirus infection after allogeneic hematopoietic stem cell transplantation.

Introduction: Cytomegalovirus (CMV) can cause various end-organ diseases in immunocompromised hosts, including allogeneic hematopoietic cell transplant (allo-HSCT) recipients. Interestingly, CMV viremia has been associated with various complications and poor prognosis in allo-HSCT recipients. Complications involving the central nervous system (CNS) occur in 9-14% of patients following allo-HSCT. However, autoimmune encephalitis (AE) secondary to CMV infection after allo-HSCT has rarely been reported. Here we report a case of possible AE following CMV viremia after allo-HSCT, which was successfully treated with high-dose pulsed methylprednisolone and intravenous immunoglobulins (IVIg). Case description: A 53-year-old female underwent allo-HSCT for T-lymphoblastic lymphoma/leukemia. The patient developed CMV viremia on day 36 after transplantation, and serum CMV-DNA remained positive after initiating ganciclovir antiviral therapy, turning negative one month later. Four months later, she started experiencing memory impairment, weakness in the left limbs, cognitive dysfunction, and hallucinations. A magnetic resonance imaging brain scan showed scattered ischemic lesions under the bilateral frontal cortex. Viral detection in cerebral spinal fluid (CSF) by next-generation gene sequencing technology showed no obvious abnormality. Antibodies specific to AE and paraneoplastic diseases in serum and CSF were absent. The oligoclonal bands in the CSF were detected using isoelectric focusing and immunofixation, and the results were negative. However, after extensive investigation regarding infections, autoimmune disorders, and recurrence of the malignancy, possible AE could not be excluded. The patient was treated with high-dose steroids combined with IVIg therapy; the patient's symptoms were significantly improved. Conclusion: The mechanisms of AE after allo-HSCT and the relationship with CMV infection should be further studied. Therefore, reporting this and similar cases will improve our awareness and understanding of the underlying disease mechanisms.

Application of quantitative contrast-enhanced ultrasound for evaluation and guiding biopsy of peripheral pulmonary lesions: a preliminary study.

AIM: To evaluate the usefulness of contrast-enhanced ultrasonography (CEUS) in differentiating malignant from benign peripheral pulmonary lesions, and to evaluate the feasibility, accuracy, and utility of CEUS-guided biopsy for peripheral pulmonary lesions. MATERIALS AND METHODS: Thirty-three patients with histopathologically confirmed peripheral pulmonary lesions (22 malignant, 11 benign) were enrolled in this retrospective study. Conventional ultrasound (US) was first performed and then CEUS with a contrast-specific mode and sulfur hexafluoride-filled microbubble contrast agent. CEUS indices-time of enhancement (TE), time to peak (TP), extent of peak (EP), mean transit time (MTT), area under curve (AUC), and slope-were recorded and compared between the groups. The ability of CEUS and US to detect necrotic areas within lesions was also compared and the accuracy of CEUS-guided biopsy was calculated. RESULTS: On CEUS, TE was significantly shorter in acute pneumonia lesions than in other types of lesions (p=0.03). Other indices were not significantly different between benign and malignant lesions. Detection of necrosis within lesions was significantly higher with CEUS than with US (51.5% versus 27.3%; p=0.04). The accuracy of CEUS-guided biopsy was 96.9% (32/33). CONCLUSION: The study findings suggest that CEUS can identify necrotic areas within lesions, and thereby, play a useful role in imaging-guided biopsy. The present pilot study indicates that CEUS may help to identify acute pneumonia lesions from other types of pulmonary lesions. CEUS might be a useful additional technique for the diagnosis of lung lesions.

Pathological changes from the originating to the peripheral sites of Sinonasal Inverted Papilloma are the underlying mechanisms of preoperative MRI-tumor origin prediction.

BACKGROUND: Our previous study showed that convoluted cerebriform pattern (CCP)-based reverse tracing method in preoperative magnetic resonance imaging (MRI) is a reliable tool in predicting originating site of sinonasal inverted papilloma (SNIP). This study aimed to determine the underlying pathological mechanism of the preoperative MRI-CCP reverse tracing method by assessing the histopathological changes from the origin to the peripheral sites of SNIP. METHODOLOGY: The originating site of SNIP was predicted by preoperative MRI in 30 consecutive patients suspected to have primary SNIP. Samples of SNIP originating and peripheral sites were processed by pathological staining for evaluation of stroma score, micro-vessel density (MVD), and tight junction proteins (claudin-5, zonula occludens (ZO)-1 and occludin) expression. RESULTS: The originating site of SNIP was accurately predicted by preoperative MRI in all patients. Stroma scores, and MVD were significantly greater in the periphery of SNIP than in the originating site. In contrast, Claudin-5 expression in micro-vessels was greater at the originating site than the periphery. CONCLUSIONS: More edematous stroma and intensive micro-vessels with defective tight junction in periphery of SNIP result in more contrast agent diffusing and CCP that can only be observed at the periphery of SNIP on T2 and contrast-enhanced T1 weighted MR images, which may be the mechanisms underlying the CCP reverse tracing method.

Removal of heavy metal chromium using cross-linked chitosan composite nanofiber mats.

Uniform chitosan composite nanofiber mats (CS/PAAS) containing 4.0 wt% polyacrylic acid sodium loading have been prepared by electrospinning, followed by annealing at elevated temperature to improve solvent resistance and mechanical strength. The CS/PAAS nanofiber mats have been characterized by scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FT-IR) and tensile strength analysis. The prepared nanofiber mats have been shown to be much better than the pristine chitosan powder to adsorb the chromium (VI) ion. SEM-Energy dispersive X-ray spectroscopic (SEM-EDS) and positron annihilation lifetime spectroscopic (PALS) analysis show that Cr(VI) ion can freely permeate into the composite nanofiber and coordinate with the internal chitosan chain molecules. The adsorption abilities of these cross-linked CS/PAAS nanofiber mats are dependent on the chitosan contents as well as N-atom basicity of the chitosan chain. The adsorption capacity toward Cr(VI) ion was improved to 78.92 mg/g after modification of the chelating ligands for the cross-linked CS/PAAS composite nanofibers.

Co-biodegradation of anthracene and naphthalene by the bacterium Acinetobacter johnsonii.

NAP (Naphthalene) and ANT (anthracene) usually co-exist in environment and possessed interactional effects on their biodegradation in environment. Presently, a strain of Acinetobacter johnsonii was employed to degrade NAP and ANT in single- and dual-substrate systems. NAP was utilized as prefer substrate by cells to accelerate ANT biodegradation. As much as 200 mg L-1 ANT could be entirely degraded with 1,500 mg L-1 NAP, which was beyond bacterial potential in single substrate system. Especially, the shortest biodegradation period (103 h) for ANT was observed with the presence of 50 mg L-1 NAP. By contrast, ANT showed strong inhibition on NAP degradation, while the peak biodegradation of 1,950 mg L-1 NAP with 50 mg L-1 ANT could still proceed. By introducing an inhibition constant parameter to fit the inhibition on cells, modeling indicated the substrate inhibition for NAP and ANT over the concentrations of 174 and 49 mg L-1, respectively. Furthermore, enzyme assay revealed the pathway of meta fission in NAP biodegradation due to the appearance of catechol 2,3-dioxygenase activity, and low-level lipase excretion was also found in both NAP and ANT biodegradation, but hardly affect NAP and ANT biodegradation in the present study. To research the interplay of NAP and ANT is conducive to targeted decontamination.

Cockroach is a major cross-reactive allergen source in shrimp-sensitized rural children in southern China.

BACKGROUND: Little is known about the prevalence of food allergy (FA) in China. The aim of this study was to investigate the disparity of FA between urban and rural areas in southern China. METHODS: EuroPrevall questionnaire responses were obtained from 5542 school-age children in urban Guangzhou and 5319 in rural Shaoguan. A case-control study enrolled 190 children with adverse reactions (ARs) after food intake as cases and 212 controls in Guangzhou, whereas 116 cases and 233 controls in Shaoguan. These subjects underwent skin prick test (SPT) and serum IgE measurements to food and inhalant allergens. Allergen extracts from shrimp, house dust mite (HDM), and cockroach were prepared for IgE cross-reactivity testing in 23 Guangzhou and 20 Shaoguan shrimp-sensitized subjects. RESULTS: The prevalence of ARs to shrimp was higher in Guangzhou than in Shaoguan children (3.5% vs 1.4%, P < .001). However, sensitization rate to shrimp (SPT: 3.7% vs 11.2%, P = .015; IgE: 12.6% vs 36.2%, P < .001) and cockroach (SPT: 5.3% vs 33.5%; IgE: 2.6% vs 27.6%, P < .001) was lower in Guangzhou. A significant correlation between shrimp and HDM/cockroach IgE was found in Shaoguan children. The proportions of positive IgE to tropomyosin (Pen a 1, Der p 10) were lower than 7.4% in both areas. Cockroach allergen has a significantly higher inhibition rate of binding to IgE to house dust mite allergens in Shaoguan sera. CONCLUSION: Shrimp is a common allergic food in southern China. Higher proportion of shrimp sensitization in rural subjects could be explained by cross-reactivity to cockroach. Tropomyosin was not a major allergen responding to the cross-reactivity.

[Clinicopathologic characteristics of plasma cell myeloma with marrow fibrosis].

Objective: To study the clinicopathologic features of plasma cell myeloma(PCM) with bone marrow fibrosis (MF). Methods: The clinicopathologic data of 175 cases of newly diagnosed PCM patients were retrospectively analyzed. Based on reticular fiber staining, these cases were divided into PCM-MF and non-PCM-MF groups. Results: Sixty-three cases were PCM-MF(36%), 112 were non-PCM-MF (64%). No statistical difference in gender, age, hemoglobin level, platelet counts, the classification of immunoglobulin, ISS staging, immunohistochemical phenotypes and genetic features was found between PCM-MF and non-PCM-MF groups (P>0.05). Compared to non-PCM-MF group, lactate dehydrogenase (LDH)level and renal impairmentrate were higher in PCM-MF group (P<0.05). The degree of bone marrow hyperplasia, the percentage of myeloma cells and cells with plasmablastic morphology were significantly higher in PCM-MF group(P<0.05). Conclusion: The higher LDH level, renal impairment rate, and more significant bone marrow hyperplasia, proliferation of plasma cells and plasmablastic myeloma cells infiltration indicate poor prognosis of PCM-MF patients.

Cl-Amidine Prevents Histone 3 Citrullination and Neutrophil Extracellular Trap Formation, and Improves Survival in a Murine Sepsis Model.

Sepsis refers to the presence of a serious infection that correlates with systemic and uncontrolled immune activation. Posttranslational histone modification plays an important role in chromatin decondensation, which is regulated by citrullination. Citrullinated histone H3 (H3cit) has been identified as a component of neutrophil extracellular traps (NETs), which are released into the extracellular space as part of the neutrophil response to infection. The conversion of arginine to citrulline residues on histones is catalyzed by peptidylarginine deiminase 4 (PAD4). This study's goals were to characterize the presence of PAD4-catalyzed H3cit and NET formation during the onset of sepsis and elucidate the effects on the immune response when this mechanism of action is blocked. Adult C57BL/6 male mice were treated with Cl-amidine, an inhibitor of PAD4, 1 h prior to sepsis induced by cecal ligation and puncture (CLP). Twenty-four hours after CLP, cytokine levels, H3cit protein expression, neutrophil counts, and NET production were evaluated in the peritoneal cavity. Survival studies were also performed. Here we demonstrate that Cl-amidine treatment prior to CLP improves overall survival in sepsis and the abrogation of PAD4 has minimal effects on the proinflammatory immune response to sepsis, while it has no effect on overall neutrophil migration to the peritoneum.

Consequences of extracellular trap formation in sepsis.

PURPOSE OF REVIEW: This review will focus on in-vivo findings derived from animal models of sepsis regarding the trapping role of neutrophil extracellular traps (NETs) which is difficult to assess ex vivo. The NETotic response of neutrophils at sites of sterile injury or autoimmune disease is destructive as no antimicrobial advantage to the host is realized and dampening NETosis is largely beneficial. In early stages of local infection or in sepsis, the trapping function of NETs may help abscess formation and limit microbial dissemination. RECENT FINDINGS: The trapping function of NETs limits bacterial dissemination keeping an abscess from becoming bacteremic or confining tissue infection to local sites. Once containment is lost and disease has progressed, the best therapeutic approach suggested by animal studies to date is to inhibit protein arginine deiminase 4 and prevent NETosis rather than attempting to neutralize caustic NET components. Prognostic value may best be realized by taking cell free DNA, citrulllinated histones, neutrophil function and counts of immature granulocytes into consideration rather than rely on any one measure alone. SUMMARY: The trapping function of NETs may supercede the value of antimicrobial function in the early phases of sepsis such that degradation of the DNA backbone is contraindicated.

Integrin Cross-Talk Regulates the Human Neutrophil Response to Fungal ß-Glucan in the Context of the Extracellular Matrix: A Prominent Role for VLA3 in the Antifungal Response.

Candida albicans infection produces elongated hyphae resistant to phagocytic clearance compelling alternative neutrophil effector mechanisms to destroy these physically large microbial structures. Additionally, all tissue-based neutrophilic responses to fungal infections necessitate contact with the extracellular matrix (ECM). Neutrophils undergo a rapid, ECM-dependent mechanism of homotypic aggregation and NETosis in response to C. albicans mediated by the ß2 integrin, complement receptor 3 (CR3, CD11b/CD18, αMß2). Neither homotypic aggregation nor NETosis occurs when human neutrophils are exposed either to immobilized fungal ß-glucan or to C. albicans hyphae without ECM. The current study provides a mechanistic basis to explain how matrix controls the antifungal effector functions of neutrophils under conditions that preclude phagocytosis. We show that CR3 ligation initiates a complex mechanism of integrin cross-talk resulting in differential regulation of the ß1 integrins VLA3 (α3ß1) and VLA5 (α5ß1). These ß1 integrins control distinct antifungal effector functions in response to either fungal ß-glucan or C. albicans hyphae and fibronectin, with VLA3 inducing homotypic aggregation and VLA5 regulating NETosis. These integrin-dependent effector functions are controlled temporally whereby VLA5 and CR3 induce rapid, focal NETosis early after binding fibronectin and ß-glucan. Within minutes, CR3 undergoes inside-out auto-activation that drives the downregulation of VLA5 and the upregulation of VLA3 to support neutrophil swarming and aggregation. Forcing VLA5 to remain in the activated state permits NETosis but prevents homotypic aggregation. Therefore, CR3 serves as a master regulator during the antifungal neutrophil response, controlling the affinity states of two different ß1 integrins, which in turn elicit distinct effector functions.

Neutrophil Integrins and Matrix Ligands and NET Release.

Neutrophils are motile and responsive to tissue injury and infection. As neutrophils emigrate from the bloodstream and migrate toward a site of affliction, they encounter the tissue extracellular matrix (ECM) and thereby engage integrins. Our laboratory studies the neutrophilic response to the fungal pathogen Candida albicans either in the filamentous state of the microbe or to the purified pathogen-associated molecular pattern, ß-glucan. We have gained an appreciation for the role of integrins in regulating the neutrophil anti-Candida response and how the presence or absence of ECM can drive experimental outcome. The ß2 integrin CR3 (complement receptor 3; αMß2; Mac-1; CD11b/CD18) plays an important role in fungal recognition by its ability to bind ß-glucan at a unique lectin-like domain. The presence of ECM differentially regulates essential neutrophil anti-fungal functions, including chemotaxis, respiratory burst, homotypic aggregation, and the release of neutrophil extracellular traps (NETs). We have shown that NET release to C. albicans hyphae or immobilized ß-glucan occurs rapidly and without the requirement for respiratory burst on ECM. This is in contrast to the more frequently reported mechanisms of NETosis to other pathogens without the context of ECM, which occur after a prolonged lag period and require respiratory burst. As expected for an ECM-dependent phenotype, NETosis and other neutrophil functions are dependent on specific integrins. The focus of this review is the role of ECM ligation by neutrophil integrins as it pertains to host defense functions with an emphasis on lessons we have learned studying the anti-Candida response of human neutrophils.

NETosis in Neonates: Evidence of a Reactive Oxygen Species-Independent Pathway in Response to Fungal Challenge.

Release of neutrophil extracellular traps (NETs) is a significant antimicrobial host defense mechanism in adults. In neonates, fungal sepsis is a frequent cause of morbidity and mortality and may be a consequence of inadequate neutrophil defense functions. Like neutrophils from adult donors, we found that neutrophils from neonates formed robust cellular aggregates and released NETs in response to fungal ß-glucan and Candida albicans hyphae when presented with extracellular matrix. Therefore, in response to fungal stimulation, neonatal neutrophils are capable of NETosis. Neonate susceptibility to fungal infections may not be due to an inability of their neutrophils to produce NETs.

Aggravation of airway inflammation and hyper-responsiveness following nasal challenge with Dermatophagoides pteronyssinus in perennial allergic rhinitis without symptoms of asthma.

BACKGROUND: House dust mites are the most prevalent allergen causing sensitizations in patients with rhinitis and asthma in China. We aimed to investigate the changes in both upper and lower airway inflammation and responsiveness following Dermatophagoides pteronyssinus (Der-p) nasal provocation test (NPT) in rhinitis patients. METHODS: Study subjects included 15 nonasthmatic Der-p-sensitized rhinitis (AR) patients with airway hyper-responsiveness (AHR) (AR+AHR+), 15 AR patients without AHR (AR+AHR-), 15 healthy controls (HCs) with Der-p sensitization (HC+DP+), and 15 HC without Der-p sensitization (HC+DP-). All subjects underwent Der-p NPT. Visual analogue scale (VAS) scores of nasal symptoms, nasal lavage and nasal airway resistance (NAR) measurement, sputum induction, and forced expiratory volume in 1 second (FEV1 ) were performed. Airway responsiveness to histamine bronchoprovocation (PD20 -FEV1 ) and exhaled nitric oxide (FeNO) was determined. RESULTS: NAR increased significantly in all subjects with the greatest effect seen in AR+AHR+ individuals. VAS increased in all subjects at 30 min and returned to baseline at 6 h, with significantly higher levels in AR+AHR+ and AR+AHR- subjects (P < 0.05). Eosinophils in nasal lavage fluid and sputum increased significantly after NPT in AR+AHR+ and AR+AHR- subjects (P < 0.001). FEV1 % and PD20 -FEV1 decreased and FeNO increased significantly after NPT only in AR+AHR+ subjects (P < 0.05). Nasal lavage eosinophil count was positively correlated with sputum eosinophil count and the level of FeNO and negatively correlated with FEV1 and PD20 . CONCLUSIONS: House dust mite nasal provocation test induces and aggravates both upper and lower airway inflammation and hyper-responsiveness in patients with persistent allergic rhinitis without asthmatic symptoms.

Describing directional cell migration with a characteristic directionality time.

Many cell types can bias their direction of locomotion by coupling to external cues. Characteristics such as how fast a cell migrates and the directedness of its migration path can be quantified to provide metrics that determine which biochemical and biomechanical factors affect directional cell migration, and by how much. To be useful, these metrics must be reproducible from one experimental setting to another. However, most are not reproducible because their numerical values depend on technical parameters like sampling interval and measurement error. To address the need for a reproducible metric, we analytically derive a metric called directionality time, the minimum observation time required to identify motion as directionally biased. We show that the corresponding fit function is applicable to a variety of ergodic, directionally biased motions. A motion is ergodic when the underlying dynamical properties such as speed or directional bias do not change over time. Measuring the directionality of nonergodic motion is less straightforward but we also show how this class of motion can be analyzed. Simulations are used to show the robustness of directionality time measurements and its decoupling from measurement errors. As a practical example, we demonstrate the measurement of directionality time, step-by-step, on noisy, nonergodic trajectories of chemotactic neutrophils. Because of its inherent generality, directionality time ought to be useful for characterizing a broad range of motions including intracellular transport, cell motility, and animal migration.

Ceramide-activated phosphatase mediates fatty acid-induced endothelial VEGF resistance and impaired angiogenesis.

Endothelial dysfunction, including endothelial hyporesponsiveness to prototypical angiogenic growth factors and eNOS agonists, underlies vascular pathology in many dysmetabolic states. We investigated effects of a saturated free fatty acid, palmitic acid (PA), on endothelial cell responses to VEGF. PA-pretreated endothelial cells had markedly diminished Akt, eNOS, and ERK activation responses to VEGF, despite normal VEGFR2 phosphorylation. PA inhibited VEGF-induced angiogenic cord formation in Matrigel, and PA-treated endothelial cells accumulated early species (C16) ceramide. The serine palmitoyltransferase inhibitor myriocin reversed these defects. Protein phosphatase 2A (PP2A) became more eNOS-associated in PA-treated cells; the PP2A inhibitor okadaic acid reversed PA-induced signaling defects. Mice fed a diet high in saturated fat for 2 to 3 weeks had impaired i) aortic Akt and eNOS phosphorylation to infused VEGF, ii) ear angiogenic responses to intradermal adenoviral-VEGF injection, and iii) vascular flow recovery to hindlimb ischemia as indicated by laser Doppler and αVß3 SPECT imaging. High-fat feeding did not impair VEGF-induced signaling or angiogenic responses in mice with reduced serine palmitoyltransferase expression. Thus, de novo ceramide synthesis is required for these detrimental PA effects. The findings demonstrate an endothelial VEGF resistance mechanism conferred by PA, which comprises ceramide-induced, PP2A-mediated dephosphorylation of critical activation sites on enzymes central to vascular homeostasis and angiogenesis. This study defines potential molecular targets for preservation of endothelial function in metabolic syndrome.

Technical advance: introducing a novel metric, directionality time, to quantify human neutrophil chemotaxis as a function of matrix composition and stiffness.

A direct consequence of cellular movement and navigation, migration incorporates elements of speed, direction, and persistence of motion. Current techniques to parameterize the trajectory of a chemotaxing cell most commonly pair migration speed with some measure of persistence by calculating MSD, RMS speed, TAD, and/or CI. We address inherent limitations in TAD and CI for comparative analysis by introducing two new analytical tools to quantify persistence: directionality index and directionality time. With the use of these tools, we show that the mechanical properties of the underlying substrate contribute significantly to the regulation of human neutrophil chemotaxis toward fMLP on Fgn-, Col-, and Fn-coated gels of varying elasticity. The ß1-integrin ligand Col demonstrated mechanosensitive speed. In contrast, ß2-integrin ligand Fgn supported mechanosensitive persistence. Fn, recognized by ß1 and ß2 integrins, mechanoregulated speed and persistence. Blocking ß2 integrins of cells migrating on Fn identified an underlying ß2-integrin-directed modulation of persistence. These data demonstrate that individual components of the neutrophil chemotactic response show integrin dependence and are finely tunable with different ligand, mechanotactic, and chemotactic cues, underscoring the need for sensitive analytical methods.

An extracellular matrix-based mechanism of rapid neutrophil extracellular trap formation in response to Candida albicans.

The armament of neutrophil-mediated host defense against pathogens includes the extrusion of a lattice of DNA and microbicidal enzymes known as neutrophil extracellular traps (NETs). The receptor/ligand interactions and intracellular signaling mechanisms responsible for elaborating NETs were determined for the response to Candida albicans. Because the host response of extravasated neutrophils to mycotic infections within tissues necessitates contact with extracellular matrix, this study also identified a novel and significant regulatory role for the ubiquitous matrix component fibronectin (Fn) in NET release. We report that recognition of purified fungal pathogen-associated molecular pattern ß-glucan by human neutrophils causes rapid (≤ 30 min) homotypic aggregation and NET release by a mechanism that requires Fn. Alone, immobilized ß-glucan induces reactive oxygen species (ROS) production but not NET release, whereas in the context of Fn, ROS production is suppressed and NETs are extruded. NET release to Fn with ß-glucan is robust, accounting for 17.2 ± 3.4% of total DNA in the cell population. Release is dependent on ß-glucan recognition by complement receptor 3 (CD11b/CD18), but not Dectin-1, or ROS. The process of NET release included filling of intracellular vesicles with nuclear material that was eventually extruded. We identify a role for ERK in homotypic aggregation and NET release. NET formation to C. albicans hyphae was also found to depend on ß-glucan recognition by complement receptor 3, require Fn and ERK but not ROS, and result in hyphal destruction. We report a new regulatory mechanism of NETosis in which the extracellular matrix is a key component of the rapid antifungal response.

Association of IL-1ß and IL-6 gene polymorphisms with recurrent spontaneous abortion in a Chinese Han population.

As certain cytokines may play a role in unexplained recurrent spontaneous abortion (RSA) and also some cytokine gene polymorphisms may affect the level of cytokine production, the aim of this study was to investigate the relationship between Chinese RSA and polymorphisms of the genes coding for interleukin (IL)-1ß (-31C/T, -511C/T, +3954C/T) and IL-6 (-634C/G). Women (n = 162) with at least three consecutive spontaneous abortions and 156 ethnically matched healthy women with at least one successful pregnancy were included. Genotypes were determined using restriction fragment length polymorphism analysis of polymerase chain reaction products. No significant differences were found in the IL-1ß-31T, -511T and +3954T distributions between the RSA group and the control group. On the other hand, the frequencies of the IL-6-634GG genotype and -634G allele were significantly decreased in the RSA group versus the control group (genotype: P = 0.0003; allele: P = 0.002), suggesting the IL-6-634C/G polymorphism might be a possible genetic protective factor for RSA.

Lectin site ligation of CR3 induces conformational changes and signaling.

Neutrophils provide an innate immune response to tissues infected with fungal pathogens such as Candida albicans. This response is tightly regulated in part through the interaction of integrins with extracellular matrix ligands that are distributed within infected tissues. The ß(2) integrin, CR3 (CD11b/CD18), is unique among integrins in containing a lectin-like domain that binds the fungal pathogen-associated molecular pattern ß-glucan and serves as the dominant receptor for recognition of fungal pathogens by human granulocytes. ß-Glucan, when isolated in soluble form, has been shown to be a safe and effective immune potentiator when administered therapeutically. Currently a pharmaceutical grade preparation of ß-glucan is in several clinical trials with an anti-cancer indication. CR3 binding of extracellular matrix, carbohydrate, or both ligands simultaneously differentially regulates neutrophil function through a mechanism not clearly understood. Using FRET reporters, we interrogated the effects of soluble ß-glucan on intracellular and extracellular CR3 structure. Although the canonical CR3 ligand fibrinogen induced full activation, ß-glucan alone or in conjunction with fibrinogen stabilized an intermediate conformation with moderate headpiece extension and full cytoplasmic tail separation. A set of phosphopeptides differentially regulated by ß-glucan in a CR3-dependent manner were identified using functional proteomics and found to be enriched for signaling molecules and proteins involved in transcriptional regulation, mRNA processing, and alternative splicing. These data confirm that CR3 is a signaling pattern recognition receptor for ß-glucan and represent the first direct evidence of soluble ß-glucan binding and affecting a signaling-competent intermediate CR3 conformation on living cells.