Expression Levels Of Myeloperoxidase Research Articles

D-mannose alleviates chronic periodontitis in rats by regulating the functions of neutrophils

BackgroundPeriodontitis is a chronic inflammatory disease characterized by the destruction of the components of the periodontium. It significantly impacts oral health and has been linked to systemic conditions like cardiovascular disease and diabetes. The critical role of neutrophils in the occurrence and development of chronic periodontitis has been paid increasing attention. The study aimed to explore the protective effects of D-mannose on chronic periodontitis and determine whether its underlying mechanisms is related to neutrophils.MethodsTo explore the protective effects of D-mannose on chronic periodontitis, the eight-week-old Sprague Dawley rat model of lipopolysaccharide (LPS)-induced periodontitis was established, followed by D-mannose treatment by oral gavage. To evaluate the protective effects of D-mannose against periodontal bone loss, methylene blue staining, hematoxylin and eosin (H&E) staining, and micro-CT scanning were utilized. Then, immunofluorescence (IF), Western Blot, and RT-PCR were applied to assess the expression levels of pro-inflammatory cytokines (IL-1β, IL-6, and IL-17), anti-inflammatory cytokine (IL-10), tumor necrosis factor-alpha (TNF-α), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), ten-eleven translocation 2 (TET2), and key glycolytic enzymes (HK1, HK2, PFKFB3), and to examine D-mannose’s impact on the recruitment and activation of neutrophils in the gingiva. Additionally, neutrophils isolated from the peripheral blood of healthy rats were treated with LPS and D-mannose, and changes in the expression levels of myeloperoxidase (MPO), IL-1β, IL-6, IL-17, IL-10, and TET2 were observed via IF.ResultsIn vivo, D-mannose inhibited LPS-induced alveolar bone resorption in rats. After D-mannose treatment, the expression levels of IL-17 (p<0.01) and TET2 (p<0.01) were suppressed by IF, and the expression levels of IL-1β (p<0.05), IL-17 (p<0.05) and TET2 (p<0.01) were downregulated by WB. The results of qPCR showed that D-mannose reduced the expression levels of IL-1β (p<0.05), IL-6 (p<0.01), IL-17 (p<0.01), TNF-α (p<0.01), G-CSF (p<0.01), GM-CSF (p<0.01), TET2 (p<0.01), HK1 (p<0.01), HK2 (p<0.01), and PFKFB3 (p<0.01). D-mannose also inhibited the recruitment and activation of neutrophils in LPS-treated rat gingival tissues. In vitro, the results of IF showed that D-mannose inhibited the activation of neutrophils stimulated by LPS, downregulated the expression of IL-1β (p < 0.05), IL-6, IL-17 (p < 0.01), and TET2 (p < 0.01), and upregulated the expression of IL-10 (p < 0.01).ConclusionsD-mannose can alleviate chronic periodontitis in rats by regulating the functions of neutrophils, potentially associated with the expression of TET2 and glycolysis, providing new insights into the potential application of D-mannose to chronic periodontitis.

Immunohistochemical expression of netosis markers (MPO, H2A, H2B) in placenta of RPL women in Basrah Province

Background. Recurrent Pregnancy Loss (RPL) is a multifactorial disorder significantly impacting women’s health globally. Emerging research has implicated Neutrophil Extracellular Traps (NETs) in the pathophysi­ology of RPL, suggesting an immunological underpinning to miscarriage occurrences. Aim. The study aim to evaluate the role and expression of NETosis markers (MPO, histones H2A, H2B) in women with RPL in comparison to pregnant women. Methods. This prospective cohort study, conducted from January 2022 to July 2023, included 30 participants (20 RPL patients and 10 controls) from Al Basrah Maternity and Child Hospital. Immunohistochemical technique were employed to detect Myeloperoxidase and histones in placental samples. Statistical analysis was performed using SPSS, with significance set at p&lt;90.05. Results. Immunohistochemical analysis of placental tissues indicated significant overexpression of Myelo­peroxidase (MPO), and histones H2A and H2B, in RPL patients compared to control, emphasizing the potential involvement of NETs in placental pathology associated with miscarriage. The expression levels of MPO were positive in 80% of RPL patients but undetected in control (p = 0.0001). Histone expressions also differed significantly, with H2A detected in 55% of RPL patients compared to none in control (p = 0.001) and H2B in 70% versus 30% (both p &lt;0.001).

Quantitative proteomic analysis of circulating exosomes reveals the mechanism by which Triptolide protects against collagen-induced arthritis.

Triptolide (TP), a natural product derived from the herbal medicine Tripterygium wilfordii, exhibits potent immunosuppressive activity. However, the mechanisms underlying its effects in rheumatoid arthritisremain incompletely understood. Collagen-induced arthritis (CIA) model was induced in Sprague-Dawley rats by immunization with bovine type II collagen, and TP was administrated as treatment. The therapeutic effect of TP was evaluated based on paw swelling, histopathology, and serum levels of inflammatory factors. Exosomes isolated from rat serum were characterized by transmission electron microscopy, dynamic light scattering, and western blotanalysis. Proteomic profiling of exosomes was analyzed by direct DIA quantitative proteomics analysis. Gene ontology and the Kyoto Encyclopedia of Genes and Genomes databases were employed for enrichment analysis related to molecular function, biological processes, and signaling pathways. Western blot analysis was used to analyze differentially expressed proteins. TP treatment ameliorated arthritic phenotypes in CIA rats as evidenced by reduced arthritis score, paw swelling, pathological injury severity scores, and serum levels of inflammatory cytokines. The proteomic analysis revealed that TP treatment significantly inhibited complement and coagulation cascades, interleukin-17 signaling pathway, and cholesterol metabolism, which were reactivated in CIA rats. Importantly, lipocalin 2 (LCN2) and myeloperoxidase (MPO) levels were markedly upregulated in the CIA group but suppressed upon TP administration. Furthermore, in synovial tissues, LCN2 and MPO expression levels were also elevated in the CIA group but decreased following TP treatment. Our findings demonstrate that TP alleviates CIA, possibly through modulation of exosomal LCN2 and MPO proteins.

Investigation of inflammatory mechanisms induced by croton oil in mouse ear

Croton oil is liquid at room temperature, with a pale-yellow color and spicy odor. It is commonly used in combination with phenol as a chemical peeling agent in dermatology, which reveals its caustic exfoliating effects. Topical use of croton oil at a high dose produces skin irritation, inflammation, swelling, pain, and even tumors. Therefore, croton oil has been widely used for inflammation, pain, and tumor related research, with different animal models having been established. However, mechanistic studies through which croton oil induces skin swelling, injury and activates tissue repair/regeneration are limited. The present study used croton oil to induce mouse ear edema and examined tissue responses 4 h after exposure. To this end, croton oil was applied to the ventral side of mouse ears, followed by tissue collection. Samples were analyzed by hematoxylin and eosin (H&E) staining, toluidine blue staining, and immunohistochemistry staining for myeloperoxidase (MPO) and matrix metalloproteinase-9 (MMP-9). Western blotting and ELISA were also carried out for MMP-9 together with unbiased proteomic analysis using mass-spectrometry. Results from our study demonstrated that as soon as 4 h of exposure to 2.5 % croton oil, the expression levels of MPO and MMP-9 in the dermis significantly increased compared to acetone-treated (vehicle) control ears, as did other inflammatory reactions such as swelling and neutrophil aggregation and infiltration. Subsequently, proteomic analysis confirmed that croton oil treatment resulted in significant upregulation of proteins such as myeloperoxidase (MPO), matrix metalloproteinase-9 (MMP-9), and matrix metalloproteinase-8 (MMP-8) in the ear skin. Interestingly, mouse ears treated with acetone vehicle showed differential expression of 2,478 proteins relative to naïve tissues; among those differentially expressed in acetone-treated samples were members of the phosphatidylinositol-glycan biosynthesis class N, T and U proteins (PIGN, PIGT, and PIGU). Overall, this work confirms the presence of neutrophil-derived MPO and MMP-9 and extends the body of knowledge to show that MMP-8 is also present during croton oil-mediated skin inflammation in the mouse ear; moreover, we find that acetone vehicle is not inert and has effects on the skin that should be considered moving forward.

Hypobaric Hypoxia Aggravates Renal Injury by Inducing the Formation of Neutrophil Extracellular Traps through the NF-κB Signaling Pathway

The hypersensitivity of the kidney makes it susceptible to hypoxia injury. The involvement of neutrophil extracellular traps (NETs) in renal injury resulting from hypobaric hypoxia (HH) has not been reported. In this study, we aimed to investigate the expression of NETs in renal injury induced by HH and the possible underlying mechanism. A total of 24 SD male rats were divided into three groups (n=8 each): normal control group, hypoxia group and hypoxia+pyrrolidine dithiocarbamate (PDTC) group. Rats in hypoxia group and hypoxia+PDTC group were placed in animal chambers with HH which was caused by simulating the altitude at 7000 meters (oxygen partial pressure about 6.9 kPa) for 7 days. PDTC was administered at a dose of 100 mg/kg intraperitoneally once daily for 7 days. Pathological changes of the rat renal tissues were observed under a light microscope; the levels of serum creatinine (SCr), blood urea nitrogen (BUN), cell-free DNA (cf-DNA) and reactive oxygen species (ROS) were measured; the expression levels of myeloperoxidase (MPO), citrullinated histone H3 (cit-H3), B-cell lymphoma 2 (Bcl-2), Bax, nuclear factor kappa B (NF-κB) p65 and phospho-NF-κB p65 (p-NF-κB p65) in rat renal tissues were detected by qRT-qPCR and Western blotting; the localization of NF-κB p65 expression in rat renal tissues was observed by immunofluorescence staining and the expression changes of NETs in rat renal tissues were detected by multiplex fluorescence immunohistochemical staining. After hypoxia, the expression of NF-κB protein in renal tissues was significantly increased, the levels of SCr, BUN, cf-DNA and ROS in serum were significantly increased, the formation of NETs in renal tissues was significantly increased, and a large number of tubular dilatation and lymphocyte infiltration were observed in renal tissues. When PDTC was used to inhibit NF-κB activation, NETs formation in renal tissue was significantly decreased, the expression level of Bcl-2 in renal tissues was significantly increased, the expression level of Bax was significantly decreased, and renal injury was significantly alleviated. HH induces the formation of NETs through the NF-κB signaling pathway, and it promotes apoptosis and aggravates renal injury by decreasing Bcl-2 and increasing Bax expression.

Porous COS@SiO2 Nanocomposites Ameliorate Severe Acute Pancreatitis and Associated Lung Injury by Regulating the Nrf2 Signaling Pathway in Mice.

Severe acute pancreatitis (SAP) is associated with high rates of mortality and morbidity. Chitosan oligosaccharides (COSs) are agents with antioxidant properties. We developed porous COS@SiO2 nanocomposites to study the protective effects and mechanisms of COS nanomedicine for the treatment of acute pancreatitis. Porous COS@SiO2 nanocomposites released COSs slowly under pH control, enabling sustained release and maintaining the drug at a higher concentration. This study aimed to determine whether porous COS@SiO2 nanocomposites ameliorate SAP and associated lung injury. The SAP model was established in male C57BL/6 mice by intraperitoneal injection of caerulein. The expression levels of myeloperoxidase, malondialdehyde, superoxide dismutase, nuclear factor-kappa B (NF-κB), the NOD-like receptor protein 3 (NLRP3) inflammasome, nuclear factor E2-related factor 2 (Nrf2), and inflammatory cytokines were detected, and a histological analysis of mouse pancreatic and lung tissues was performed. In the SAP groups, systemic inflammation and oxidative stress occurred, and pathological damage to the pancreas and lung was obvious. Combined with porous COS@SiO2 nanocomposites before treatment, the systemic inflammatory response was obviously reduced, as were oxidative stress indicators in targeted tissues. It was found that Nrf2 was significantly activated in the COS@SiO2 treatment group, and the expressions of NF-κB and the NLRP3 inflammasome were notably decreased. In addition, this protective effect was significantly weakened when Nrf2 signaling was inhibited by ML385. This demonstrated that porous COS@SiO2 nanocomposites activate the Nrf2 signaling pathway to inhibit oxidative stress and reduce the expression of NF-κB and the NLRP3 inflammasome and the release of inflammatory factors, thus blocking the systemic inflammatory response and ultimately ameliorating SAP and associated lung injury.

Effect of Shaoyao Tang on ulcerative colitis in rats via regulation of TLR4/NF-κB signal pathway

The aim of this paper was to investigate the effect of Shaoyao Tang on ulcerative colitis(UC) in rats via regulation of TLR4/NF-κB signal pathway. A total of 56 Wistar rats were randomly divided into 6 groups: normal control group(double distilled water), model group(double distilled water), mesalazine group(10 mL·kg~(-1)), high dose, middle dose and low dose Shaoyao Tang groups(2.4, 1.2, and 0.6 g·mL~(-1)). After UC rat models were established by 2, 4-dinitrochlorobenzene(DNCB)/ethanol enema, the rats received double distilled water or corresponding drugs twice a day for 7 days. After the treatment cycle, the general performance and disease activity index(DAI) of rats were observed on the next day. Then the rats were sacrificed. The length of colon was measured. Macroscopic and histological score of colon were evaluated. Histopathological changes of colon were observed by HE staining. Ultraviolet spectrophotometry detection was used to detect the content of myeloperoxidase(MPO) in blood and colon tissues. The levels of P-selectin, macrophage migration inhibitory factor(MIF) and thromboxane B_2(TXB_2) in blood and colon tissues were determined by ELISA. Immunohistochemistry and Western blot analysis were performed to detect the protein expressions of TLR4 and NF-κB in colon tissues. The results showed that as compared with the model group, Shaoyao Tang of different doses improved the general performance of UC rats. Moreover, high-dose Shaoyao Tang group showed the most obvious effect in scoring of disease activity index(P<0.001); both medium and high doses of Shaoyao Tang significantly inhibited the colon shortening and pathological injury, with significantly decreased expression levels of MPO, P-selectin, MIF and TXB_(2 )in serum and colon tissues of UC rats(P<0.001). Immunohistochemistry and Western blot assay showed that the levels of TLR4 and NF-κB protein expression in the colon tissues of Shaoyao Tang high-dose group were remarkably lower than that in the model group(P<0.001). This study shows that Shaoyao Tang has protective and repairing effects on UC, and its possible mechanism is achieved probably by regulating the TLR4/NF-κB pathway and inhibiting the expressions of MPO, P-selectin, MIF and TXB_2.

Effect of Sheng-jiang powder on multiple-organ inflammatory injury in acute pancreatitis in rats fed a high-fat diet.

BACKGROUNDObesity worsens inflammatory organ injury in acute pancreatitis (AP), but there is no effective preventive strategy. Sheng-jiang powder (SJP) has been shown to alleviate multiple-organ inflammatory injury in rats with high-fat diet-induced obesity. Hence, SJP is supposed to have an effect on multiple-organ inflammatory injury in AP in rats fed a high-fat diet.AIMTo explore how obesity may contribute to aggravating inflammatory organ injury in AP in rats and observe the effect of SJP on multiple-organ inflammatory injury in AP in rats fed a high-fat diet.METHODSRats were randomly assigned to a control group (CG), an obese group (OG), and an SJP treatment group (SG), with eight rats per group. The rats in the OG and SG were fed a high-fat diet. From the third week, the rats in the SG were given oral doses of SJP (5 g/kg of body weight). After 12 wk, AP was induced in the three groups. Serum amylase level, body weight, Lee’s index, serum biochemistry parameters, and serum inflammatory cytokine and tissue cytokine levels were assessed, and the tissue histopathological scores were evaluated and compared.RESULTSCompared with the CG, serum triglyceride, total cholesterol, interleukin-6, and interleukin-10 levels were significantly higher in the OG, and serum high-density lipoprotein cholesterol level was significantly lower in the OG. Moreover, enhanced oxidative damage was observed in the pancreas, heart, spleen, lung, intestine, liver, and kidney. Evidence of an imbalanced antioxidant defense system, especially in the pancreas, spleen, and intestine, was observed in the obese AP rats. Compared with the OG, serum high-density lipoprotein cholesterol, interleukin-10, and superoxide dismutase expression levels in the pancreas, spleen, and intestine were increased in the SG. Additionally, SJP intervention led to a decrease in the following parameters: body weight; Lee’s index; serum triglyceride levels; serum total cholesterol levels; malondialdehyde expression levels in the pancreas, heart, spleen, lung, and liver; myeloperoxidase expression levels in the lung; and pathological scores in the liver.CONCLUSIONObesity may aggravate the inflammatory reaction and pathological multiple-organ injury in AP rats, and SJP may alleviate multiple-organ inflammatory injury in AP in rats fed a high-fat diet.

Expression changes of CD177 and MPO as novel biomarkers in lung tissue of CLP model rats

Sepsis is an unregulated systemic response to microbial invasion that can lead to multiple organ failure. This study aims at investigating the relationships among myeloperoxidase (MPO) and CD177 in major organ systems including whole blood, liver, and lung tissues in septic rats. Sepsis was induced by cecal ligation and puncture (CLP) in female Wistar rats. Whole blood, liver, and lung samples were obtained from rats of 3 groups (n = 10 for each group, n total = 30: control as a wild-type group, laparotomy group (LAP), and CLP). Gene expression of MPO and CD177 in targeted tissues was determined by real-time PCR after CLP. MPO activity was also determined by ELISA method for the result validation of the real-time PCR. Expression levels of MPO increased significantly in all targeted organs in the CLP group, while CD177 expression was upregulated only in lung tissue in response to sepsis (P < 0.05). The results obtained with ELISA analysis also show that MPO level was significantly increased in all the targeted tissues in the CLP group (P < 0.05). A high level of MPO as an inflammatory enzyme can be a potentially novel biomarker for sepsis in all organs. On the other hand, CD177 may be a marker in lung tissue.

Correlation between expressions of myeloperoxidase and eosinophil cationic protein in sputum and clinical features of asthma-chronic obstructive pulmonary disease overlap

To observe the role of myeloperoxidase(MPO)and eosinophilic cationic protein(ECP)in the airway inflammation and their correlation with clinical feature in asthma-COPD overlap (ACO) patients. Twenty patients with COPD, 20 with asthma, 20 with ACO and 20 control subjects underwent pulmonary function test for measurement of forced expiratory volume in 1 second (FEV1), forced vital capacity (FVC), peak expiratory flow (PEF), and maximum midexpiratory flow (MMF25/75). COPD assessment test (CAT) was used to evaluate the clinical symptoms of the patients with COPD and ACO. The asthma control test (ACT) was used to evaluate the asthma control in the patients with asthma and ACO. Induced sputum samples were collected from the subjects for analysis of neutrophil and eosinophil ratios, and enzyme-linked immunosorbent assay was used to determine the expression levels of MPO and ECP in the sputum. No significant difference was observed in the CAT scores between ACO group and COPD group (P&gt; 0.05). Compared with the asthma group, the patients with ACO had significantly lower ACT scores and lower FEV1, PEF and MMF25/75 (P &lt; 0.05). The patients with ACO had significantly higher FVC and sputum eosinophil ratio than those with COPD (P &lt; 0.05), and a higher sputum neutrophil ratio than those with asthma (P &lt; 0.01). In ACO group, the MPO level in sputum was significantly higher than that in the asthma group (P &lt; 0.05), while sputum ECP level was significantly higher than that in both the asthma group and COPD group (P &lt; 0.05 or 0.01). In ACO group, sputum MPO level was positively correlated with sputum neutrophil ratio (r=0.8358, P &lt; 0.01) but was not correlated with CAT score or FEV1 (P&gt; 0.05); sputum ECP level was positively correlated with sputum eosinophil ratio (r=0.4666, P &lt; 0.05) and was inversely correlated with ACT score (r=-0.4966, P &lt; 0.05) and FEV1 (r=-0.4610, P &lt; 0.05). Both neutrophilic and eosinophilic inflammations occur in the airway of patients with ACO, and their sputum ECP level is negatively correlated with asthma control and obstructive airflow limitation.

Association of polymorphism in P16 and myeloperoxidase genes with susceptibility to oral lesions in North Indian population

BackgroundExpression of P16 and MPO (myeloperoxidase) has been proposed as a marker for malignant transformation. In MPO, a G/A polymorphism at 463 bp upstream of the transcription start site of the enzyme has functional outcome, where G allele results in higher level of MPO expression than A allele. Protein P16, a key regulator in cell cycle, is also commonly associated with cancer. SNP of P16 exon3 (C540G) has been found to be linked with tumor aggressiveness. ObjectiveThe study aimed to investigate SNP of P16C540G and myeloperoxidase (MPO) −463G/A for their potential association in oral lesions with risk to oral squamous cell carcinoma (OSCC). MethodsIn this hospital based case control study, single nucleotide polymorphism in P16 and MPO genes was studied by polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) method in patients of oral lesions or OSCC and compared with age matched healthy controls. The test group consisted of 226 patients from both rural and urban areas whereas the control group had 103 individuals who did not show a history of oral lesions or OSCC. Genotype and allele frequencies were analyzed by chi-square test and strength of associations by odds ratio with 95% confidence intervals. ResultsThis case–control study demonstrates that people having P16C540G carrier genotypes 540 (CG/GG) were found to be protected from the development of leukoplakia (P = 0.004, <0.001, OR = 0.2451, 0.0818, 95% CI = 0.097–0.619, 0.0295–0.2272 respectively). Whereas, SNP at G463A of MPO gene was found to be not associated with the risk of oral lesions (P = 0.8351, OR = 0.5667, 95% CI = 0.0968–3.3168). ConclusionOur results suggest that single nucleotide polymorphisms in P16 genes may affect the susceptibility to oral lesions; while genotype of MPO showed no association with the development of oral lesions.

Association of myeloperoxidase with ovarian cancer

Myeloperoxidase (MPO) is an oxidant generating enzyme normally restricted to myeloid cells, however aberrant MPO expression has been found to occur in non-myeloid cells in some disease states. The functional -463GA promoter polymorphism alters MPO expression levels. The -463G is within an SP1 binding site and is associated with higher gene expression. The G allele is most frequent with ~62% of European populations being GG homozygotes. The GA polymorphism has been associated with risk or survival in a variety of cancers including lung and breast cancer. In this study we determined the frequency of the -463G/A polymorphism in 230 ovarian cancer patients, 75 patients with borderline ovarian tumors, and 299 healthy controls. The GG genotype was found to be overrepresented in patients with early stage ovarian cancer (83.3% GG, p = 0.008) as compared to healthy controls (62% GG), suggesting that MPO oxidants may increase risk. Immunohistochemical analysis revealed MPO expression in a subset of columnar ovarian epithelial carcinoma cells in early stage carcinomas.

Neutrophil dysfunction induced by hyperglycemia: modulation of myeloperoxidase activity

Our data suggest that impaired activity of myeloperoxidase (MPO) may play an important role in the dysfunction of neutrophils from hyperglycemic rats. Neutrophil biochemical pathways include the NADPH oxidase system and the MPO enzyme. They both play important role in the killing function of neutrophils. The effect of hyperglycemia on the activity of these enzymes and the consequences with regard to Candida albicans phagocytosis and the microbicidal property of rat peritoneal neutrophils is evaluated here. The NADPH oxidase system activity was measured using chemiluminescence and cytochrome C reduction assays. MPO activity was measured by monitoring HOCl production, and MPO protein expression was analysed using Western blot and immunofluorescence. C. albicans phagocytosis and death were evaluated by optical microscopy using the May-Grunwald-Giemsa staining method. ROS generation kinetic was slightly delayed in the diabetic group. MPO expression levels were higher in diabetic neutrophils; however, MPO activity was decreased in these same neutrophils compared with the controls. C. albicans phagocytosis and killing were lower in the diabetic neutrophils. Based on our experimental model, the phagocytic and killing functions of neutrophil phagocytosis are impaired in diabetic rats because of the decreased production of HOCl, highlighting the importance of MPO in the microbicidal function of neutrophils.

Level of Myeloperoxidase Expression as a Crucial Determinant of Sensitivity of Myeloid Leukemia Cells to Parthenolide-Induced Apoptosis

Parthenolide (PTL) is a sesquiterpene lactone found as the major active component in Feverfew (Tanacetum parthenium). PTL is a strong inhibitor of NF-kB activation and STAT transcriptional activity, resulting in a downregulation of antiapoptotic gene transcription. Recently, PTL was demonstrated to have promising anti-cancer effects through inhibition of DNA synthesis and reactive oxygen species (ROS)-associated intrinsic apoptosis. However, the sensitivity to PTL-induced cell death was different according to leukemia cells. Therefore, it will be important to identify parameters predicting response to PTL-induced cell death. Myeloperoxidase (MPO), a typical lineage marker for acute myeloid leukemia (AML), was also shown to have a prognostic significance in this disorder. Since it was shown that MPO is a potential regulator of oxidative stress, we examined the effect of MPO expression upon the PTL-induced leukemia cell death. First we compared the extent and mechanism of PTL-induced cell death between parental K562 and MPO-overexpressing K562 (K562/MPO) cells. K562/MPO cells were kindly provided by Dr. Sawayama (Nagasaki University, Japan). Annexin V labeling evaluation revealed that PTL induced apoptosis in K562/MPO cells in a dosedependent manner. The fraction of apoptotic cells after 24 hours treatment with 10mM of PTL was significantly higher in K562/MPO cells (52.2 ± 0.4%) compared to parental K562 cells (13.1 ± 4.8%, p<0.001). When the cells were treated with PTL, the population that lost mitochondrial membrane disruption (MMP) was 45.1 ± 12.4% in K562/MPO cells, which was significantly higher than K562 cells (0.7 ± 0.1%, p<0.001). Cleavage of caspase-3, -8, -9, and PARP was observed in K562/MPO cells after PTL treatment, whereas it was not shown in K562 cells. We next examined the possible involvement of ROS in PTL-induced cell death by flow cytometric analysis using dihydroethidium fluorescent probe. PTL drastically increased relative ROS levels in K562/MPO cells (4.1 ± 0.1). However, the increase in ROS levels induced by PTL was not demonstrated in K562 cells (1.5 ± 0.1, p<0.01). Marked downregulation of Bcl-2, Bcl-xL, and NF-kB was demonstrated preferentially in K562/MPO cells. c-Jun N-terminal kinase phosphorylation was remarkably increased only in K562/MPO cells. We next evaluated the PTL-induced cell death in primary leukemic blasts obtained from patients with AML. Quantitative measurement of MPO expression was done using flow cytometry analysis. Interestingly, the fraction of apoptotic cells induced by 24 hours treatment of PTL was significantly higher in AML specimens consisting of higher than 50% of MPO-positive cells (MPOhi cases; 44.5 ± 1.6%, n = 6) compared to AML specimens consisting of lower than 20% of MPO-positive cells (MPOlo cases; 1.4 ± 1.1%, n = 4, p<0.001). Our findings indicate that PTL induces apoptosis in myeloid leukemia cells is associated with increased ROS and the extent of MPO expression is a crucial determinant of their sensitivity to PTL-induced cell death.

Circulating Myeloperoxidase (MPO) as a Tumor Marker in Patients with Chronic Myeloid Leukemia (CML).

MPO is a hemoprotein expressed in polymorphonuclear neutrophils and their precursors. Free circulating MPO in the plasma or serum is an important indicator of inflammation in various inflammatory diseases, whereas cellular expression levels of MPO, assessed by cytochemical staining, are used extensively for the diagnosis and subtyping of various leukemias. The role of circulating MPO levels in the evaluation of CML has not been assessed. In this study, we examined the association of plasma MPO levels with hematologic characteristics and clinical outcome in 107 patients with Philadelphia chromosome-positive CML: 74 in chronic phase (CP) and 33 in accelerated/blastic (ACC/BC) phase). 100 apparently healthy individuals were included as a control group. All patients received imatinib mesylate therapy, with a median follow-up period of 18.6 months (range, 2.1–31.1 months). Overall, MPO levels did not differ significantly between CP and ACC/BC patients (Wilcoxon test, P=0.34). However, both CP (median, 38.2; range, 4.9–11421.6 ng/mL) and ACC/BC (median, 47.00; range, 3.4–1423.6 ng/mL) groups had significantly higher levels than control subjects (median, 4.9; range, 3.5–20.6 ng/mL). Response rates were 77% for CP patients and 57% for ACC/CP patients. When both groups were considered together, nonresponders had significantly higher MPO levels than responders (P = 0.03). In separate analyses, this association held true for ACC/BC patients (P = 0.04) but not for the CP group. MPO levels correlated strongly with WBC, LDH, percent basophiles, and percent Philadelphia chromosome-positive metaphases (all P <0.001). These data suggest that plasma MPO levels are elevated in CML and may be useful in predicting response to imatinib mesylate therapy for patients in ACC/BC phase; whether the association of MPO levels with response reflects an underlying association with tumor burden requires further study. Longer follow-up with larger samples sizes may be needed to accurately evaluate the relationship between plasma MPO levels with response and overall survival for patients in CP.

Clinical Relevance of Circulating Myeloperoxidase (MPO) in Acute Myeloid Leukemia (AML) and Myelodysplastic Syndrome (MDS).

MPO, a hemoprotein expressed in polymorphonuclear neutrophils and their precursors, is an important component of the antimicrobial activity of phagocytes and is associated with inflammatory response. Cellular expression levels of MPO, assessed by cytochemical staining, are used extensively for the diagnosis and subtyping of various leukemias. However, the association of circulating (plasma or serum) MPO levels with hematologic values and clinical outcomes of AML and MDS has not been reported. We investigated whether MPO, presumably released from leukemic cells, can be detected in the plasma of patients with AML and MDS. We further assessed the association of plasma MPO levels with hematologic characteristics and outcomes. 144 patients with AML, 28 patients with MDS, and 100 control subjects were included in the analysis. Patients with AML (median 19.3, range 1.0–9514.7 ng/mL) and MDS (median 13.6, range 1.0–3021.9 ng/mL) had significantly higher plasma MPO levels than control subjects (median, 4.9; range, 3.5–20.6 ng/mL). Because MPO levels and overall survival did not differ significantly between AML and MDS patients, the 2 groups were considered together in subsequent analyses. MPO levels were inversely correlated with overall survival (Cox regression analysis, P<0.001). In the multivariate model, the inverse correlation between MPO levels and survival was independent of cytogenetics, performance status, anticident hematologic disease (AHD), age, and diagnosis (AML vs MDS). Response to therapy in AML and MDS patients was not associated with plasma MPO levels. However, responders with high MPO levels had shorter remission duration (CRD), although this difference did not reach statistical significance (P = 0.07). As expected, MPO levels correlated with the level of differentiation of leukemic cells (FAB subclassification): patients with M0 and M6 had lower MPO levels than those with M2, M3, or M4 (P = 0.0001). MPO levels also correlated positively with white cell count, beta2-microglobulin, monocyte, LDH, BUN, and creatinine levels. These data indicate that plasma MPO levels are elevated in AML and MDS and may be useful as a prognostic factor. Our findings also raise the question of how the proinflammatory properties of MPO might affect the clinical course of these diseases.

−463 G/A Myeloperoxidase Promoter Polymorphism Is Associated with Clinical Manifestations and the Course of Disease in MPO-ANCA-Associated Vasculitis

Wegener's granulomatosis, microscopic polyangiitis, and Churg Strauss syndrome are forms of systemic vasculitides in which neutrophils and monocyte macrophages infiltrate the walls of small blood vessels, leading to destruction and occlusion. These diseases are associated with autoantibodies directed against granular components of neutrophils and monocytes, i.e., antineutrophil cytoplasmic antibodies (ANCA). The most common target antigens of ANCA in these vasculitides are myeloperoxidase (MPO) and proteinase 3 (PR3). ANCA-stimulated neutrophils injure endothelial cells, a process that is dependent upon the production of reactive oxygen radicals and the release of granular components such as MPO and PR3. Here we investigate whether a common functional MPO promoter polymorphism (−463 G/A) is associated with increased incidence and clinical aspects of ANCA-associated small vessel vasculitis. Genotyping was carried out for 142 patients with ANCA-associated small vessel vasculitis and 129 ethnically matched controls. The GG genotype was found to be associated with an increased risk for MPO-ANCA-associated vasculitis in females (86% GG, P = 0.045), but not males (64% GG, P = 1.0). Interestingly, the MPO A allele is associated with an increased incidence of relapses (P = 0.012) and an earlier age at diagnosis (P = 0.03) of MPO-ANCA-associated vasculitis. Both these associations are specific for MPO-ANCA and are not observed in patients with PR3-ANCA-associated vasculitis. These findings suggest that MPO expression levels influence the disease course of MPO-ANCA-associated vasculitis and further support the view that genetic factors are involved in the pathophysiology of this autoimmune disease.

A functional myeloperoxidase polymorphic variant is associated with coronary artery disease in French-Canadians

Background One of the enzymes involved in the production of free radicals in atherosclerotic plaques is myeloperoxidase (MPO). There is a functional G/A polymorphism 463 bp upstream of the transcription start site of the enzyme with the G allele associated with a higher level of MPO expression than the A allele. Considering the potential role of MPO in the process of atherosclerosis, studying the relationship between this polymorphism and the incidence of coronary artery disease (CAD) seems reasonable. Method We performed a case-control study. The case group consisted of 229 patients who had angiographically proved atherosclerotic plaques in their coronary arteries. The control group consisted of 217 individuals who did not have a history of coronary artery disease, stroke, or peripheral vascular disease. Results We found that allele A of the MPO gene was less frequent in cases with CAD. In a recessive model patients with the AA genotype had a decreased risk of CAD (odds ratio 0.138, 95% confidence interval 0.040-0.474). In a dominant model a significant protective role for AA or AG versus GG was also detected (odds ratio 0.639, 95% confidence interval 0.436-0.937). Conclusion Our findings suggest that the –463 G/A polymorphism of the MPO gene influences the risk of CAD. This effect may be mediated by the effect of this polymorphism on the transcription level of the MPO gene. (Am Heart J 2001; 142:336-9.)

Involvement of STAT3 in the Granulocyte Colony-stimulating Factor-induced Differentiation of Myeloid Cells

Granulocyte colony-stimulating factor (G-CSF) stimulates proliferation and differentiation of the progenitor cells of neutrophilic granulocytes. The binding of G-CSF to its receptor specifically activates JAK1 and JAK2 kinases, as well as STAT3, a signal transducer and activator of transcription (STAT). To examine the role of STAT3 in G-CSF receptor-mediated signal transduction, two different forms of the dominant negative STAT3 were introduced into a mouse myeloid cell line that exogenously expresses the mouse G-CSF receptor. In response to G-CSF, the parental myeloid cells grew for about 4 days, and then they stopped dividing and differentiated into cells with lobulated nuclei. During this period, the expression of the myeloperoxidase (MPO) gene was induced, while c-myc gene expression was down-regulated. In contrast, in the cells expressing the dominant negative STAT3, G-CSF could induce neither growth arrest nor morphological change. However, the induction of the MPO gene by G-CSF was not affected by the dominant negative STAT3. These results indicate that STAT3 activation is responsible for part of the G-CSF-induced differentiation of neutrophils but that another pathway, involving the expression of the MPO gene, that does not utilize the activated STAT3, is also required to fully differentiate the cells.

Human Myeloperoxidase Gene: Molecular Cloning and Expression in Leukemic Cells

We have molecularly cloned the human myeloperoxidase (MPO) gene from the lambda gt11 expression library by screening with an affinity-purified MPO antibody. The cDNA clone of the MPO gene was used to study MPO gene expression in leukemic cells. The amino acid sequence predicted from the nucleotide sequence of the cDNA clone pMP401 matched exactly the 23 amino acid sequence of the NH2-terminal of the 60,000 MPO subunit. We found that MPO cDNA hybridized to a single EcoRI genomic band of 19 kb, indicating that the MPO gene represents a single gene in the human genome. Northern blot analysis of RNA isolated from leukemic cell lines and acute myelogenous leukemia (AML) patients' samples shows that MPO gene expression correlated with myeloid lineage. The intensity of MPO mRNA expression on Northern blot correlated with the level of MPO expression by cytochemical staining. Multiple species of MPO mRNA were found. This indicates that a single MPO gene may encode different RNA species through a mechanism of posttranscriptional processing or that multiple transcriptional start/termination sites exist in the MPO gene.