Matrix Metalloproteinase Activity in Pediatric Acute Lung Injury

Pediatric Acute Lung Injury (ALI) is associated with a high mortality and morbidity, and dysregulation of matrix metalloproteinases (MMPs) may play an important role in the pathogenesis and evolution of ALI. Here we examined MMP expression and activity in pediatric ALI compared with controls. MMP-8, -9, and to a lesser extent, MMP-2, -3, -11 and -12 were identified at higher levels in lung secretions of pediatric ALI patients compared with controls. Tissue Inhibitor of Matrix metalloproteinase-1 (TIMP-1), a natural inhibitor of MMPs was detected in most ALI samples, but MMP-9:TIMP-1 ratios were high relative to controls. In subjects who remained intubated for ≥10 days, MMP-9 activity decreased, with > 80% found in the latent form. In contrast, almost all MMP-8 detected at later disease course was constitutively active. Discriminating MMP-9:TIMP-1 ratios were found in those who had a prolonged ALI course. These results identify a specific repertoire of MMP isoforms in the lung secretions of pediatric ALI patients, and demonstrate inverse changes in MMPs -8 and -9 with protracted disease.     

Differentiation of Human Monocytes in Vitro Following Exposure to Canova in the Absence of Cytokines

Canova is an immunomodulatory, homeopathic preparation that has been shown to activate macrophages in vitro and in vivo, with resultant enhanced spreading of the cells and formation of microvillus extensions from the cell body. Since monocytes are the precursor cells of macrophages and dendritic cells, the objective of the current study was to investigate the effects of Canova on the differentiation of human blood monocytes in vitro. Monocytes were isolated, grown in culture, and exposed to 10 and 20% Canova without the addition of cytokines. After 48?h, monocytes were prepared for analysis by scanning electron microscopy, while cells kept in culture for 7 days and exposed to Canova on days 1, 3, and 4 were analyzed by flow cytometry for alterations in the levels of expression of CD1a, CD11c, CD14, CD80, CD83, CD86, and HLA-DR. SEM revealed that monocytes exposed to 10% Canova had a morphological appearance similar to that of macrophages. Various cytoplasmic projections were observed with pseudopodia formation. Flow cytometric analysis after exposure of monocytes to 10 and 20% Canova indicated high cell viability and upregulation of CD80, compatible with differentiation into either macrophages or dendritic cells. Exposure to Canova per se causes activation of monocytes with resultant differentiation into large macrophage-like cells of indeterminate phenotype that have increased expression of CD80. Like cytokines, Canova induces differentiation of monocytes, an activity that may underpin the immunomodulatory activity of this product.     

血清基质金属蛋白酶活性与动脉粥样硬化斑块稳定性的实验观察

目的观察血清MMP-2、MMP-9在不同时期的兔动脉粥样硬化动物模型中的变化,研究其影响因素及与斑块稳定性的关系。方法将30只长耳白兔随机分为3组:普通饮食组(n=10),高脂饮食组(n=10),球囊损伤 高脂饮食组(n=10),3个月后以中国斑点蝰蛇毒和组胺触发使斑块破裂。其间监测血脂情况,并行血清MMP-2、MMP-9检测。动物处死后查找动脉硬化斑块行病理检测。结果普通饮食组实验中各检测项目均无明显差异。高脂饮食及球囊损伤 高脂饮食组一个月后血脂明显升高。药物触发后高脂饮食组血清MMP-9活性明显增加,球囊损伤 高脂饮食组血清MMP-2、MMP-9活性均明显增加。结论在兔动脉粥样硬化的动物模型中,斑块处于不稳定状态时血清MMP-9活性明显增加,而MMP-2的活性升高可能与内皮机械损伤有关。     

Cytokine production in peripheral blood cells during and outside the pollen season in birch-allergic patients and non-allergic controls

BACKGROUND: The naturally occurring pollen season permits observation of the kinetic changes in the process of allergic inflammation. We examined cytokine production in peripheral blood (PB) T cells and monocytes obtained from birch-allergic patients both during and outside the pollen season. METHODS: PB from 16 patients and six healthy controls was obtained during the alder pollen season, at the beginning and the peak of the birch pollen season and outside the pollen season. Mononuclear cells (MNC) were stimulated with allergen and polyclonal activators. For flow cytometric analysis, MNC were stained with monoclonal antibodies (MoAbs) against the cell surface markers CD3, CD8, CD14 and the intracellular cytokines IL-4, IL-5, IL-10, IL-12, IL-13, granulocyte macrophage-colony stimulating factor (GM-CSF) and IFN-gamma. RESULTS: In allergic patients, significant increases in clinical symptoms, use of medication, eosinophil numbers and birch-specific IgE were found during the pollen season. In vitro allergen stimulation increased the number of GM-CSF+ monocytes (P<0.01) and this increase was dependent on allergen exposure. The IL-4/IFN-gamma ratio rose (P<0.001) at the peak of birch pollen season and the ratio correlated with symptom scores during the birch season. In the CD4+ cell population, the numbers of GM-CSF+ cells were higher throughout the alder and birch seasons compared with outside the pollen season (P<0.05). No such changes were seen in the healthy controls. CONCLUSIONS: The main finding of our study was the increased percentage of GM-CSF+ monocytes in atopic subjects compared with healthy controls. In allergic patients, natural seasonal pollen exposure resulted in increased numbers of GM-CSF+ cells among both monocytes and CD4+ T cells. We have also shown that a seasonal change in Th2/Th1 cytokine ratio requires an adequate and prolonged allergen stimulation that is seen late in the pollen season.     

Tension Force Downregulates Matrix Metalloproteinase Expression and Upregulates the Expression of Their Inhibitors through MAPK Signaling Pathways in MC3T3-E1 cells

Objective: Matrix metalloproteinases (MMPs), produced by osteoblasts, catalyze the turnover of extracellular matrix (ECM) molecules in osteoid, and the regulation of MMP activity depends on interactions between MMPs and tissue inhibitors of metalloproteinases (TIMPs). We focused on the degradation process of ECM in osteoid that was exposed to mechanical strain, and conducted an in vitro study using MC3T3-E1 osteoblastic cells to examine the effects of tension force (TF) on the expression of MMPs and TIMPs, and activation of mitogen-activated protein kinase (MAPK) pathways.Design: Cells were incubated on flexible-bottomed culture plates and stimulated with or without cyclic TF for 24 hours. The expression of MMPs and TIMPs was examined at mRNA and protein levels by real-time RT-PCR and Western blotting, respectively. The phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, p38 MAPK, and stress-activated protein kinases/c-jun N-terminal kinases (SAPK/JNK) were examined by Western blotting.Results: TF decreased the expression of MMP-1, -3, -13 and phosphorylated ERK1/2. In contrast, TF increased the expression of TIMP-2, -3 and phosphorylated SAPK/JNK. The expression of MMP-2, -14, TIMP-1, -4 and phosphorylated p38 MAPK was unaffected by TF. MMP-1, -3 and -13 expression decreased in cells treated with the ERK inhibitor PD98059 compared with untreated control cells. The JNK inhibitor SP600125 inhibited the TF-induced upregulation of TIMP-2 and -3.Conclusions: The results suggest that TF suppresses the degradation process that occurs during ECM turnover in osteoid via decreased production of MMP-1, -3 and -13, and increased production of TIMP-2 and -3 through the MAPK signaling pathways in osteoblasts.     

Clinical Differences Between Stroke and Stroke Mimics in Code Stroke Patients

BackgroundThe code stroke system is designed to identify stroke patients who may benefit from reperfusion therapy. It is essential for emergency physicians to rapidly distinguish true strokes from stroke mimics to activate code stroke. This study aimed to investigate the clinical and neurological characteristics that can be used to differentiate between stroke and stroke mimics in the emergency department (ED).MethodsWe conducted a retrospective observational study of code stroke patients in the ED from January to December 2019. The baseline characteristics and the clinical and neurological features of stroke mimics were compared with those of strokes.ResultsA total of 409 code stroke patients presented to the ED, and 125 (31%) were diagnosed with stroke mimics. The common stroke mimics were seizures (21.7%), drug toxicity (12.0%), metabolic disorders (11.2%), brain tumors (8.8%), and peripheral vertigo (7.2%). The independent predictors of stroke mimics were psychiatric disorders, dizziness, altered mental status, and seizure-like movements, while current smoking, elevated systolic blood pressure, atrial fibrillation on the initial electrocardiogram, hemiparesis as a symptom, and facial palsy as a sign suggested a stroke. In addition, the likelihood of a stroke in code stroke patients tended to increase as the number of accompanying deficits increased from the following set of seven focal neurological deficits: hemiparesis (or upper limb monoparesis), unilateral limb sensory change, facial palsy, dysarthria, aphasia (or neglect), visual field defect, and oculomotor disorder (P < 0.001).ConclusionSome clinical and neurological characteristics have been identified to help differentiate stroke mimics from true stroke. In particular, the likelihood of stroke tended to increase as the number of accompanying focal neurological deficits increased.     

Cytokine regulation of low-affinity IgE receptor (CD23) on monocytes from asthmatic subjects.

The regulation of CD23 expression (Fc epsilon RII) by cytokines on monocytes from normal subjects, asymptomatic and acute asthmatics was investigated. CD23 was weakly expressed on cells from controls, but was significantly enhanced in the two groups of asthmatics. The addition of IL-4 on monocytes induced an increase of CD23 expression in cells from controls and asthmatics. Interferon-gamma (IFN-gamma) did not modulate CD23 expression in asthmatics or control subjects, while high doses of IL-6 (2000 U/ml) enhanced CD23 expression on cells from asthmatics or controls. In vitro stimulation of monocytes with Timothy grass pollen allergen did not enhance CD23 receptor in asthmatics with a positive skin test to this pollen. We speculate that CD23 expression in asthmatics is markedly enhanced by Th2-dependent cytokines, such as IL-4 and IL-6. Thus, the regulation of Th2 cell activation by anti-cytokine therapy could have an important effect on the down-regulation of CD23 on monocytes, and in shifting a Th2 subpopulation into a Th1 subpopulation by blocking Th2-dependent cytokines.     

The Role of Raf Kinase Inhibitor Protein in Rheumatoid Fibroblast-like Synoviocytes Invasiveness and Cytokine and Matrix Metalloproteinase Expression

Fibroblast-like synoviocytes (FLS) play an important role in the pathogenesis of rheumatoid arthritis. Raf kinase inhibitor protein (RKIP) negatively regulates the Raf/MEK/ERK and NF-κB pathway. The role of RKIP in rheumatoid FLS is unknown. The purpose of the present study was to investigate the function of RKIP in rheumatoid FLS. Rheumatoid FLS were transfected with either RKIP-expressing plasmids or RKIP small interfering RNA (siRNA). RKIP protein was detected in rheumatoid synovial tissue (ST) and FLS. RKIP overexpression significantly decreased IL-6 mRNA expression in TNF-α-stimulated rheumatoid FLS. RKIP overexpression also showed a decreased trend in IL-8, MMP-1, and MMP-3 mRNA expression in TNF-α-stimulated rheumatoid FLS. RKIP silencing resulted in significantly increased MMP-1 and MMP-3 mRNA expression in TNF-α-stimulated rheumatoid FLS. RKIP silencing also increased IL-6 and IL-8 mRNA expression in TNF-α-stimulated rheumatoid FLS, but this increase did not reach statistical significance. TNF-α-induced ERK and NF-κB activation was suppressed in FLS with RKIP overexpression. RKIP silencing resulted in a significantly higher invasion index in TNF-α-stimulated rheumatoid FLS compared to controls. These results suggest that RKIP might be a potential therapeutic target for rheumatoid arthritis.     

An Extract of the Medicinal Mushroom Agaricus blazei Murill Differentially Stimulates Production of Pro-inflammatory Cytokines in Human Monocytes and Human Vein Endothelial Cells in vitro

An extract of the edible mushroom Agaricus blazei Murill (AbM) has known antitumor and anti-infection properties, probably mainly by stimulating mononuclear phagocytes of the native immune system. The aim of this work was to study the effect of AbM on the production by human monocytes and human umbilical vein endothelial cells (EC) of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, TNFα), the anti-inflammatory/T regulatory cytokine IL-10 and the pro-Th1 cytokine IL-12. AbM, in concentrations from 1–15%, induced a considerable and dose-dependent increase in production of IL-8, IL-6, TNFα and IL-1β in monocyte cultures. The biosynthesis reached a plateau at a concentration of 10% of AbM, and was most pronounced for the three former cytokines. AbM did also dose-dependently stimulate EC production of IL-8,IL-6 and TNFα, but at lower levels compared with the monocytes. AbM did neither induce synthesis of cytokines IL-10 nor IL-12 in monocytes or EC. Our results demonstrate the differential effect of AbM stimulation on the magnitude of pro-inflammatory cytokines produced by monocytes and EC.     

Paternal Leukocytes Selectively Increase Secretion of IL-4 in Peripheral Blood During Normal Pregnancies: Demonstrated by a Novel One-Way MLC Measuring Cytokine Secretion

PROBLEM: It has been proposed that immune responses in normal pregnancy are Th2-like, thereby protecting the fetus and placenta from being rejected. Some studies have shown Th2-deviated systemic responses to different antigens and mitogens. The aim of this study was to demonstrate the specific T cell cytokine responses directed toward paternal histocompatibility leukocyte antigen (HLA), because this is the most prominent target for rejection of the feto-placental unit. METHOD OF STUDY: A novel one-way mixed leukocyte culture (MLC) combined with the detection of cytokine secretion with a sensitive ELISPOT assay was developed. Peripheral blood from 11 pregnant women was investigated with respect to allo-reactivity toward paternal leukocytes and pooled leukocytes from unrelated blood donors. This was done at three different occasions during pregnancy and 8 weeks after delivery. Nine age-matched non-pregnant women served as controls. RESULTS: In the second and third trimesters of pregnancy significantly larger numbers of IL-4-secreting cells (Th2) were induced by paternal leukocytes as compared to unrelated leukocytes. CONCLUSIONS: The findings indicate a selective immune deviation toward Th2, which may protect the fetus from rejection and thus may be an important homeostatic mechanism in normal pregnancies.     

Oxygen-Dependent Processes in Monocytes and Metabolic Risk Factors for Atherogenesis

Oxygen-dependent processes in peripheral blood monocytes were intensified in patients with metabolic cardiovascular syndrome. This was manifested in increased production of O(2)(*-) and NO. Among metabolic factors (cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triacylglycerols, glucose, etc.), products of glycosylation (fructosamine) and plasma triacylglycerols were most potent in modulating generation of O(2)(*-) and NO by monocytes.     

Correction of Th1-dominant Cytokine Profiles by High-dose Dexamethasone in Patients with Chronic Idiopathic Thrombocytopenic Purpura

To investigate the possible correcting of T helper (Th) cytokine profiles by high-dose dexamethasone (HD-DXM) therapy in chronic idiopathic thrombocytopenic purpura (ITP) with active disease, we determined the plasma levels of IFN-γ, IL-2, IL-4, IL-10, and TGF-β1 in 52 patients before and after oral administration of 40 mg/day DXM for four consecutive days. The cytokine levels were measured by enzyme-linked immunosorbent assay. The results showed that initial responses were reached in all patients and sustained response (SR) rate is 46.15%. The pretreatment plasma levels of both IFN-γ and IL-2 were significantly increased and those of IL-4, IL-10, and TGF-β1 significantly decreased, compared with those of the normal controls (P < 0.01), indicating a Th1-dominant cytokine profile typically found in ITP. After HD-DXM treatment, IFN-γ and IL-2 were decreased (P < 0.01), whereas IL-4 and IL-10 were increased (P < 0.05). There was no significant difference between the HD-DXM-treated patients and the normal controls (P > 0.05). TGF-β1 was also increased (P < 0.01) after HD-DXM treatment, but still lower than that of the normal controls (P < 0.05). During following-up, the cytokine profiles in the SRs remained stable compared to the posttreatment level (P > 0.05), but IFN-γ and IL-2 levels raised up, and IL-4, IL-10, and TGF-β1 levels reduced again in the relapsed patients (P < 0.01). Our data demonstrate that HD-DXM is an effective initial therapy for ITP, and the Th1 cytokine dominance could be corrected by HD-DXM.     

Matrix metalloproteinase and cytokine production by bone marrow adherent cells from multiple myeloma patients

Introduction Cultures of bone marrow stromal cells derived from the bone marrow of multiple myeloma (MM) patients were shown to exhibit several abnormalities compared with control cultures from healthy subjects. The aim of the study was to examine whether cultures of bone marrow adherent cells, at low passage level, exhibit differences in matrix metalloproteinases (MMPs) and cytokine production compared with cultures from normal donors. Materials and Methods MMP production was evaluated by gel zymography and by ELISA in supernatants of serum-free cultures of bone marrow adherent cells derived from 20 MM patients and 23 healthy controls. Spontaneous and lipopolysaccharide (LPS)- or Newcastle disease virus (NDV)-induced cytokine release was assessed in the supernatants of the cultures by the ELISA method. Results Both cultures produced MMP-1, −2, −3, and −9 under serum-free conditions; however, the levels of MMP-1 and MMP-2 were significantly higher in cultures derived from MM patients, while MMP-3 was significantly higher in control cultures. The level of MMP-9 was comparable in the cultures derived from MM patients and controls. All cultures produced interleukin (IL)-10 and IL-11 spontaneously, but after LPS or NDV induction the levels of IL-10, IL-11, interferon α, and tumor necrosis factor α, were significantly higher in the cultures derived from MM patients than in control cultures. Conclusions The results indicate that both the abnormalities in MMP production and the overproduction of cytokines (in the presence of LPS or virus, which mimic inflammatory conditions) may be involved in bone destruction and tumor spread in multiple myeloma.     

Systemic immune response in whiplash injury and ankle sprain: elevated IL-6 and IL-10

Whiplash injury and whiplash-associated disorders (WAD) are significant problems of modern society. Numerous attempts have been made to characterize the nature of whiplash injury. Whether the immune system is involved during the disease process is not known. In a prospective study, using enzyme-linked immunospot (ELISPOT) assays, we examined numbers of blood mononuclear cells (MNC) secreting pro- (IFN-gamma, TNF-alpha, IL-6) and anti-inflammatory (IL-10) cytokines in patients with WAD and, for reference, patients with ankle sprain and multiple sclerosis and healthy subjects. An immune response reflected by elevated numbers of TNF-alpha- and IL-10-secreting blood MNC was observed in patients with WAD examined within 3 days compared to 14 days after the whiplash injury. The patients with WAD examined within 3 days after the injury had also higher numbers of IL-6 and IL-10 secreting blood MNC compared to healthy subjects. The alterations of cytokine profiles observed in WAD were also observed in patients with ankle sprain when examined within 3 days after trauma. In contrast, there were no differences for cytokine profiles between patients with WAD examined 14 days after the whiplash injury and healthy subjects. Relatively minor trauma like WAD and ankle sprain are associated with a systemic dysregulation in numbers of cells secreting pro- as well as anti-inflammatory cytokines.     

Cytokine Profiles in the Central Nervous System and the Spleen During the Early Course of Experimental African Trypanosomiasis

Cytokines are important signalling proteins, which have been shown to contribute to immunopathogenesis of several inflammatory and infectious diseases such as African trypanosomiasis. The present study was conducted in order to evaluate the early induction of five potential cytokines in the central nervous system (CNS) and spleens from Trypanosoma brucei brucei (T. b. brucei)-inoculated and uninfected control Sprague-Dawley rats. In brain, choroid plexus and spleen, cytokine levels were examined by in situ hybridization and immunohistochemistry, while ELISA was used to measure cytokine levels in cerebrospinal fluid (CSF). Our results showed that interferon (IFN)-gamma and transforming growth factor (TGF)-beta were highly expressed in all compartments, but low interleukin (IL)-4, IL-10 and tumour necrosis factor (TNF)-alpha mRNA levels were registered. The pattern of these cytokines is in context with the severity of the disease because (i) IFN-gamma was previously demonstrated to promote parasite growth (ii) TNF-alpha was previously demonstrated to kill the parasites and (iii) IL-4 was previously demonstrated to promote antibody production necessary for elimination of the infection. These data support the hypothesis that cytokines may have a role in developing the disease either by enhancing the parasite growth or by suppressing the immune response.     

Cytokine-producing cells in peripheral blood of children with coeliac disease secrete cytokines with a type 1 profile.

Coeliac disease (CoD) is a small intestinal disorder characterized by crypt cell hyperplasia and villous atrophy, and the production of cytokines from T cells and macrophages are of importance for the histological changes seen in CoD. A peroral immunization with an antigen, which gives rise to a mucosal immune response, may increase the levels of circulating cytokine-producing cells, and we wanted to obtain a better picture of an eventual emergence of activated circulating T cells in the peripheral blood in children with CoD. The cytokine expression of interferon-gamma (IFN-gamma), IL-4, IL-6 and IL-10 was measured at the single-cell level by an ELISPOT method in 38 children with CoD. The numbers of IFN-gamma-producing cells in the peripheral blood was increased in children with untreated CoD (P < 0.01) and after gluten challenge (P < 0.05) compared with healthy controls. Also, the numbers of IL-6-producing cells were increased (P < 0.05) after gluten challenge compared with the healthy controls. A paired comparison showed that the numbers of IFN-gamma-producing cells increased after gluten challenge (P < 0.05), whereas no such change was seen for IL-4- or IL-10-producing cells. There were no differences in the numbers of IFN-gamma-producing cells between the group of children with treated CoD and the groups of untreated or challenged CoD children. IL-4 production correlated with serum levels of total IgE. These results show that circulating mononuclear cells in children with active CoD secrete cytokines compatible with a type 1 response.     

Neutrophils and monocytes as potentially important sources of proinflammatory cytokines in diabetes

Neutrophils and monocytes play a central role in host defence. The invading leucocytes are capable of synthesizing and releasing a variety of proinflammatory mediators including cytokines. Given the importance of cytokines in the progression of chronic and acute inflammatory processes, we aimed to ascertain whether the release of interleukin (IL)-8, IL-1beta, tumour necrosis factor (TNF)-alpha and IL-1ra of neutrophils and monocytes was modified in diabetes. To this end, we measured the release of cytokines in suspensions of cell culture in basal and lipopolysaccharide (LPS)-stimulated conditions. In basal conditions, neutrophils of diabetics release 1.6, 3.2, 1.9 and 1.9-fold higher amounts of IL-8, IL-1beta, TNF-alpha and IL-1ra, respectively, than do healthy controls. Under our experimental conditions, this effect was more evident for neutrophils than for monocytes. Incremental cytokine production was also found to occur when neutrophils were stimulated with LPS. IL-8, IL-1beta and TNF-alpha increased, respectively, by 4.0, 1.7 and 2.8-fold. Although the effect was more marked for neutrophils, monocytes showed a tendency for increased cytokine production. The discovery of this increase in cytokines released by the neutrophils of diabetics contributes towards a clearer understanding of other deficiencies described for neutrophils in diabetes, such as the migration of neutrophils to inflammatory sites, phagocytes, release of lytic proteases, production of reactive oxygen species and apoptosis. The excessive production of cytokines may lead to inappropriate activation and tissue injury and even to increased susceptibility to invasive microorganisms. Thus, the increased responsiveness of neutrophils of diabetics demonstrated in this study may be considered part of the scenario of diabetes physiopathology.