OBJECTIVETo analyze and evaluate the application of spinning disk confocal microscopy and imaging analysis software in movement and phagocytosis of neutrophils.

METHODSNeutrophils were isolated from bone marrow by centrifugation on discontinuous Percoll gradient, and then were stained with PE Gr-1 antibody and mixed with FITC-labeled Zymosan A bioparticles. Multichannel time-lapse videos were captured by using the spinning disk confocal microscopy. The result was analyzed by using volocity and ImageJ software, the parameters associated with movement and phagocytosis of neutrophils were analyzed, including morphological changes, cell tracking, pseudopod dynamics, binding and phagocytosis index.

RESULTSMost neutrophils would be polarized in response to Zymosan particles during a short time. Binding and phagocytosis process occured in forty minutes.

CONCLUSIONA method of precisely quantifying the movement and phagocytosis of neutrophils using microscopic imaging and imaging analysis technique has been set up successfully. Using this method, biological activity and function of neutrophils can be evaluated visually and rapidly. The physiologically rapid response to Zymosan particles can be applied to the neutrophils function research in the future.

Antibodies ; Bone Marrow ; Cell Movement ; Humans ; Microscopy ; Neutrophils ; Phagocytosis ; Zymosan

BACKGROUND: The immunomodulatory effects of Korean mistletoe (Viscum album Coloratum) on the innate immune responses of eel (Anguilla japonica) were studied. METHODS: Mistletoe, Freund's complete adjuvant (FCA), or phosphate-buffered saline (PBS) as a control was injected into eel peritoneal cavities. RESULTS: Nitroblue tetrazolium (NBT)-positive cells in the head kidney of eel were significantly augmented by the second day post-injection of mistletoe. Reactive oxygen intermediates (ROI) were more produced in mistletoe-injected fish kidney leucocytes than in FCA-injected ones. The level of lysozyme activity in the serum of fish 2 days after injection with mistletoe was also significantly higher than that in the serum of the control fish. The optimal concentration of mistletoe in inducing the highest serum lysozyme activity was revealed to 500microgram/200 g of fish. In phagocytic activity assay, mistletoe-sensitized eel kidney phagocytes captured more zymosan than did the control fish. CONCLUSION: Korean mistletoe appeared to be a good activator of the non-specific immune responses of eel.
Eels ; Head Kidney ; Immunity, Innate ; Kidney ; Mistletoe ; Muramidase ; Nitroblue Tetrazolium ; Oxygen ; Phagocytes ; Zymosan

Pain symptoms are a common complication of diabetic peripheral neuropathy or an inflammatory condition. In the most experiments, only one or two evident pain modalities are observed at diabetic peripheral neuropathy according to experimental conditions. Following diabetic peripheral neuropathy or inflammation, spinal glial activation may be considered as an important mediator in the development of pain. For this reason, the present study was aimed to address the induction of pain modalities and spinal glial expression after streptozotocin injection as compared with that of zymosan inflammation in the rat. Evaluation of pain behavior by either thermal or mechanical stimuli was performed at 3 weeks or 5 hours after either intravenous streptozotocin or zymosan. Degrees of pain were divided into 4 groups: severe, moderate, mild, and non-pain induction. On the mechanical allodynia test, zymosan evoked predominantly a severe type of pain, whereas streptozotocin induced a weak degree of pain (severe+moderate: 57.1%). Although zymosan did not evoke cold allodynia, streptozotocin evoked stronger pain behavior, compared with zymosan (severe+moderate: 50.0%). On the other hand, the high incidence of thermal hyperalgesia (severe+moderate: 90.0%) and mechanical hyperalgesia (severe+moderate: 85.7%) by streptozotocin was observed, as similar to that of zymosan. In the spinal cord, the increase of microglia and astrocyte was evident by streptozotocin, only microglia was activated by zymosan. Therefore, it is recommended that the selection of mechanical and thermal hyperalgesia is suitable for the evaluation of streptozotocin induced diabetic peripheral neuropathy. Moreover, spinal glial activation may be considered an important factor.
Animals ; Astrocytes ; Cold Temperature ; Hand ; Hyperalgesia ; Incidence ; Inflammation ; Microglia ; Neuroglia ; Peripheral Nervous System Diseases ; Rats ; Spinal Cord ; Streptozocin ; Zymosan

BACKGROUND/AIMS: Adenosine is an endogenous modulator of nociception. Its role in visceral nociception, particularly in visceral hyperalgesia, has not been studied. The aim of this study was to determine the effects of adenosine receptor agonists in a model of visceral hyperalgesia. METHODS: The visceromotor response (VMR) in rats to colorectal distension (CRD; 80 mmHg, 20 seconds) was quantified by electromyographic recordings from the abdominal musculature. Three hours after the intracolonic administration of zymosan (25 mg/mL, 1 mL), VMRs to CRD were measured before and after either subcutaneous or intrathecal administration of an adenosine receptor agonist. RESULTS: Subcutaneous injection of 5'-N-ethylcarboxyamidoadenosine (NECA; an A1 and A2 receptor agonist), R(-)-N6-(2-phenylisopropyl)-adenosine (R-PIA; a selective A1 receptor agonist), or CGS-21680 hydrochloride (a selective A2a receptor agonist) dose-dependently (10-100 mg/kg) attenuated the VMR to CRD, although hindlimb weakness occurred at the higher doses tested. Intrathecal administration of NECA or R-PIA dose-dependently (0.1-1.0 microgram/kg) decreased the VMR, whereas CGS-21680 hydrochloride was ineffective over the same concentration range. Higher intrathecal doses of the A1/A2 receptor agonist NECA produced motor weakness. CONCLUSIONS: Adenosine receptor agonists are antihyperalgesic, but also produce motor weakness at high doses. However, activation of the spinal A1 receptor significantly attenuates the VMR to CRD without producing motor weakness.
Adenosine ; Adenosine-5'-(N-ethylcarboxamide) ; Animals ; Hindlimb ; Hyperalgesia ; Injections, Subcutaneous ; Nociception ; Purinergic P1 Receptor Agonists ; Rats ; Receptors, Purinergic P1 ; Zymosan

Atopic dermatitis is characterized by many signs of immunodeficiency. We have performed this experiment to know whether there are reduced respiratoty burst of neutrophils in patients with atopic dermatitis in response to stimulants such as zymosan activated serum(ZAS), phorbol myristate cetate(PMA) and for- mylmethionylleucylphenylalanine(FMLP). The atopic derrqatitis group consisted of 27 patients(5 are severe, 22 are mild) and the control group consisted of 10 persons. Superoxide anion generation of neutrophils in response to stimulants was measured as nmol of reriuced cytochrome C by spectrophotometer(at 550nm, molar extinction coefficient of cytochrome C=21.lmM 1cm ). We compared the superoxide anion generation according to the severity of atopie dermatitis, total serum IgE level and eosinophil count. Results were as follows. 1. After stimulation by PMA and FMLP, superoxide anion generation in severe atopic dermatitis group decreased compared with the control and mild atopic dermatitis group. After stimulation by ZAS there was a decreasing tendency in severe atopic dermatitis group, however it was not statistically significant. 2. Superoxide anion generation had no correlation with the total serum IgE level. 3. Superoxide anion generation had no correlation with the eosinophil count. Our data suggested that some physiologic stimulants of respiratory hurst may be generated during the course of atopic dermatitis. Possible physiologic stimulants include C5a, bacterial chemotactic factors, certain arachidonate metabolites such as leukotriene B4, as well as phagocytosis. We think that these physiologic stimulants can desensitize neutrophils of atopic dermatitis in vivo specifically or onspecifically so that superoxide anion generation may be reduced in response tostimulants in vitro.
Chemotactic Factors ; Cytochromes ; Cytochromes c ; Dermatitis ; Dermatitis, Atopic* ; Eosinophils ; Humans ; Immunoglobulin E ; Leukotriene B4 ; Molar ; Myristic Acid ; Neutrophils* ; Phagocytosis ; Superoxides* ; Zymosan

Retinal pigment epithelium (RPE) constituting the outer blood-retina barrier plays an important role in ocular defense mechanism. Many studies reported that RPE participates in ongoing immune responses in the retina. However, the exact mechanism is still uncertain. Toll-like receptors (TLRs) participate in the recognition of pathogen-associated molecular patterns (PAMP), such as LPS, zymosan, lipoprotein, and dsRNA. The expression and function of TLRs in human RPE have not been established. In this study, we investigated TLRs expression in human fetal RPE and their recognition of PAMP to determine how human RPE participates in ocular defense mechanism against microbial component. RT-PCR and real time PCR revealed that TLR1 through 5 were constitutively expressed in human fetal RPE, and their expressions were slightly increased by LPS. We determined the TNF-alpha, IL-6, and IL-8 expression in human fetal RPE after treatment with LPS, zymosan, petidoglycan, or poly I:C. RT-PCR demonstrated that LPS and poly I:C treatment increased the production of TNF-alpha, IL-6, and IL-8 in human fetal RPE. LPS showed more potent effects on TNF-alpha and IL-8 production. Peptidoglycan and zymosan did not induce the production of TNF-alpha. CD14, the co-receptor of LPS was weakly expressed and functioned in recognizing LPS in human fetal RPE. These results suggest that human RPE may participate in ocular defense mechanism against microbial component through toll-like receptors.
Epithelial Cells* ; Humans* ; Interleukin-6 ; Interleukin-8 ; Lipoproteins ; Peptidoglycan ; Real-Time Polymerase Chain Reaction ; Retina ; Retinal Pigment Epithelium ; Retinaldehyde* ; Toll-Like Receptors* ; Tumor Necrosis Factor-alpha ; Zymosan

The gene expressions of Interleukin-6 (IL-6) from human peripheral blood lymphocytes (HPBL) stimulated by C. albicans were investigated by using ELISA (Enzyme linked immunosorbent assay), reverse transcription polymerase chain reaction (RT-PCR) and northern blotting. HPBL (1 X 10'/ml) obtained from normal human peripheral blood lymphocytes were cultured with live C. albicans (LCA) or heat killed C. albicans type A 311 (KCA, 3 X 10/ml) for various times (0.5, 1, 4, 8, 18, 24, 48 and 72 hours). On the purpose of this experiment, we also used lipopolysacchalide (LPS, 10 ug/ml), zymosan (1, 10, 100 ug/ml) as a polysacchaide component of the wall of yeast cells or TNFa (50, 100 ng/ml) as a IL-6 inducers. For observation of the level of IL-6 gene expression, actinomycin D (AD, 5 pg/ml) or cyclohexamide (CHX, 25 ug/ml) was added to HPBL stimulated with LCA for 0.5, 2, 4 hours and the HPBL were assessed for IL-6 mRNA. The highest value for IL-6 activity by LCA were observed at 48 hours reaction, but in the case of KCA, highest value of IL-6 activity was observed at 72 hours reaction and the value was also higher (500 pg/ml) than that of LCA (188 pg/ml). 1L-6 mRNA induced by LCA were detected up to 48 hours but in the case of KCA, the band for IL-6 mRNA were far stronger and appeared until lately than that of LCA. Therefore, the results of IL-6 gene expression agreed with that of ELISA.
Blotting, Northern ; Dactinomycin ; Enzyme-Linked Immunosorbent Assay ; Gene Expression* ; Hot Temperature ; Humans* ; Interleukin-6 ; Lymphocytes* ; Polymerase Chain Reaction ; Reverse Transcription ; RNA, Messenger ; Yeasts ; Zymosan

Dectin-1, which specifically recognizes beta-glucan of fungal cell walls, is a non-Toll-like receptor (TLR) pattern recognition receptor and a representative of C-type lectin receptors (CLRs). The importance of Dectin-1 in innate immune cells, such as dendritic cells and macrophages, has previously been well studied. However, the function of Dectin-1 in B cells is very poorly understood. To determine the role of Dectin-1 in B cell activation, we first investigated whether mouse B cells express Dectin-1 and then assessed the effect of Dectin-1 stimulation on B cell proliferation and antibody production. Mouse B cells express mRNAs encoding CLRs, including Dectin-1, and surface Dectin-1 was expressed in B cells of C57BL/6 rather than BALB/c strain. Dectin-1 agonists, heat-killed Candida albicans (HKCA) and heat-killed Saccharomyces cerevisiae (HKSC), alone induced B cell proliferation but not antibody production. Interestingly, HKSC, HKCA, and depleted zymosan (a selective Dectin-1 agonist) selectively enhanced LPS-driven IgG1 production. Taken together, these results suggest that, during fungal infection, beta-glucan-stimulated Dectin-1 may cooperate with TLR4 to specifically enhance IgG1 production by mouse B cells.
Animals ; Antibody Formation ; B-Lymphocytes* ; Candida albicans ; Cell Proliferation ; Cell Wall ; Dendritic Cells ; Immunoglobulin G* ; Lectins, C-Type ; Macrophages ; Mice* ; RNA, Messenger ; Saccharomyces cerevisiae ; Sprains and Strains ; Zymosan

OBJECTIVETo observe the regularity of change in high mobility group protein box 1 (HMGB1) content in serum and spleen of mice with multiple organ dysfunction syndrome (MODS), to analyze the correlation between HMGB1 content and major histocompatibility complex (MHC)-II---I-A(b) expression on monocytes in blood and spleen, and to explore the effect of HMGB1 on immune function of circulating monocytes and splenocytes.

METHODSOne hundred 8-week-old male 57BL/6 mice were randomly divided into normal group and experimental group subdivided into 8 subgroups: 3, 8, 12 hours, 1, 2, 3, 5-7 days and 10-12 days post zymosan injection (PZI). MODS model was replicated by injecting zymosan into the peritoneal cavity. At each time point, blood and spleen were collected to detect HMGB1 content and the rate of I-A(b) positive monocytes.

RESULTSIn normal and PZI 3-hour, 8-hour mice, serum HMGB1 was not detected, but it significantly increased at PZI 12 hours. In spleen of normal mice, there was low level of HMGB1 expression. In zymosan-treated mice, HMGB1 started to rise in spleen at PZI 3 hours. Subsequently, HMGB1 content in both serum and spleen significantly increased, and it reached the peak level in 1-2 days, decreased in 5 days, and then increased in 10-12 days. The number of I-A(b) positive monocytes in circulating blood and spleen decreased at 1-2 days (t equal to 9.589, 4.432, P <0.01) and 10-12 days following the challenge, forming a two trough like decrease, just corresponding with two-peak increase of HMGB1. However, at 3 hours after zymosan challenge, I-A(b) expression on circulating monocytes was downregulated (t =5.977, P less than 0.01), while that in spleen upregulated (t equal to 4.814, P less than 0.01).

CONCLUSIONIn mice with MODS, up-regulated HMGB1 expression can regulate I-A(b)expression on monocytes to depress their ability of presenting antigen, which results in immune disturbance contributing development of MODS.

Animals ; HMGB1 Protein ; analysis ; Histocompatibility Antigens Class II ; analysis ; Male ; Mice ; Mice, Inbred C57BL ; Monocytes ; immunology ; Multiple Organ Failure ; immunology ; Spleen ; immunology ; Zymosan ; pharmacology

Periodontitis is generally a chronic disorder characterized by breakdown of tooth-supporting tissues, producing dentition loss. Porphyromonas gingivalis (P. gingivalis), a Gram-negative anaerobic rod, is one of the major pathogens associated with periodontitis. Neutrophils are first line defense cells in the oral cavity that play a significant role in inflammatory response. Xylitol is a known anti-caries agent and has anti-inflammatory effects. In this study, we conducted experiments to evaluate anti-inflammatory effects of xylitol on P. gingivalis infected neutrophils for possible usage in prevention and treatment of periodontal infections. P. gingivalis was intraperitoneally injected and peritoneal lavage was collected for cytokine determination. For in vitro study, neutrophils were collected from mouse peritoneal cells after zymosan injection or bone marrow cells. Neutrophils were stimulated with live P. gingivalis and ELISA was used to determine the effect of xylitol on P. gingivalis induced cytokine production. IL-1β, IL-6, TNF-α concentration and neutrophil population in the peritoneal lavage was increased in P. gingivalis-infected mouse. Peritoneal cells infected with live P. gingivalis revealed significantly increased production of IL-1β, IL-6 and TNF-α at multiplicity of infection of 10. Neutrophils from bone marrow and peritoneal lavage revealed increased production of IL-1β, IL-6 and TNF-α. Xylitol significantly mitigated P. gingivalis induced cytokine production in neutrophils. Findings indicate that xylitol is an anti-inflammatory agent in neutrophils infected with live P. gingivalis, that suggests its use in periodontitis management.
Animals ; Bone Marrow ; Bone Marrow Cells ; Dentition ; Enzyme-Linked Immunosorbent Assay ; In Vitro Techniques ; Inflammation ; Interleukin-6 ; Mice ; Mouth ; Neutrophils ; Periodontitis ; Peritoneal Lavage ; Porphyromonas gingivalis ; Porphyromonas ; Xylitol ; Zymosan