Mouse MRP8 and MRP14, two intracellular calcium-binding proteins associated with the development of the myeloid lineage.

MRP8 and MRP14 are two S100-like calcium-binding proteins of unknown function, associated with numbers of human inflammatory disorders. Both molecules have been described as L1 complex, cystic fibrosis antigen, or p8 and p14. We report here the cloning of mouse MRP8 and MRP14 and their pattern of expression during hematopoiesis. Mouse MRP8 and MRP14 proteins share 59% identity with their human counterparts, but they are more divergent than the other members of the S100 protein family. Mouse MRP proteins are coexpressed in fetal myeloid progenitors, where they are detected as early as day 11 of gestation. In fetal liver and yolk sac, MRP+ cell populations increased in number, in association with the development of the myeloid lineage. In adult mouse, we identified MRP8 and MRP14 proteins in immature myeloid cells of the bone marrow, myeloid cells in the splenic red pulp and marginal zone, in addition to monocytes and blood neutrophils. However, MRP expression is lost as cells terminally differentiate into tissue macrophages. In addition, using thioglycollate-induced peritoneal inflammatory exudates, we showed that MRP8 and MRP14 proteins are highly expressed in recruited neutrophils and monocytes.     

Expression of myeloid related proteins (MRP) 8 and 14 and the MRP8/14 heterodimer in rheumatoid arthritis synovial membrane

OBJECTIVE: Myeloid related proteins (MRP) 8 and 14 and the heterodimer MRP8/14 are myeloid differentiation markers present on infiltrating tissue macrophages in inflammation but not on resident tissue macrophages. We determined the pattern of expression of MRP8, MRP14, and the MRP8/14 heterodimer (27E10 antigen) in rheumatoid arthritis (RA) synovial membrane (SM). METHODS: SM samples were obtained from patients with RA at joint replacement surgery or at arthroscopy and patients without joint disease (healthy subjects) and immunostained for MRP8, MRP14, 27E10 antigen (MRP8/14 heterodimer), and CD68. Positive cell staining was measured by quantitative analysis. RESULTS: SM from 8 patients with RA, including 7 who had paired samples from both adjacent to the cartilage-pannus junction (CPJ) and an area remote from the CPJ, and 2 healthy controls were analyzed. In RA, CD68+ cells accumulated in greater numbers adjacent to the CPJ than remote from the CPJ [mean +/- standard error of the mean (SEM) 488+/-103 and 286+/-76 cells/mm2, respectively; p = 0.01]. SM lining layer (LL) MRP8, MRP14, and 27E10 staining was observed predominantly adjacent to the CPJ and only in patients with active disease. Minimal or absent LL MRP staining was observed in non-CPJ sections. Synovial sublining layer (SL) MRP8, MRP14, and 27E10 staining was also observed only in patients with active disease, but in contrast to the LL, SL staining was observed predominantly in sections remote from the CPJ. CONCLUSION: MRP antigens, representing activation markers on SM macrophages, were observed predominantly in the LL of sections adjacent to the CPJ samples. These original observations suggest altered activation and differentiation of lining layer macrophages at the site of maximal cartilage destruction in RA.     

Lysosomes are associated with microtubules and not with intermediate filaments in cultured fibroblasts.

Double immunofluorescent labeling experiments for lysosomes and either microtubules or vimentin intermediate filaments in cultured well-spread fibroblasts show a remarkable degree of superposition of the lysosomes and the microtubules. Under two different sets of conditions where the microtubules and intermediate filaments are well segregated from one another, the lysosomes remain codistributed with the microtubules. It is suggested that this specific association of lysosomes with microtubules reflects some type(s) of linkage(s) between them and that such linkages may play an important role in the location and intracellular transport of lysosomes inside cells.     

Mitochondria are associated with microtubules and not with intermediate filaments in cultured fibroblasts.

Triple-immunofluorescence experiments with antibodies to cytochrome c oxidase, tubulin, and vimentin have been used to immunolabel the mitochondria, microtubules, and intermediate filaments inside the same cultured fibroblasts. In particular, fibroblasts were immunolabeled after they had either been transformed by infection with Rous sarcoma virus or given long-term treatment with cycloheximide. These treatments induced redistribution of the intermediate filaments into a perinuclear arrangement, segregated away from the microtubules, which remained extended to the cell periphery. In such cells, many labeled mitochondria were observed to be codistributed with the peripherally located microtubules. From these results, we infer that an association, probably involving some type of chemical linkage(s), between mitochondria and microtubules exists in these cells that is independent of the intermediate filaments.     

Elevated levels of serum MRP8/14 in ankylosing spondylitis: associated with peripheral arthritis and active disease

Clinical Rheumatology - Monocytes of patients with ankylosing spondylitis (AS) have toll-like receptor 4 (TLR4) overexpression on their monocytes. Myeloid-related protein (MRP) 8/14 protein...     

Trough imatinib plasma levels are associated with both cytogenetic and molecular responses to standard-dose imatinib in chronic myeloid leukemia

Using high-performance liquid chromatography-tandem mass spectrometry, we assessed trough imatinib plasma levels in 68 patients with chronic myeloid leukemia (CML) who responded or not to standard-dose imatinib, after at least 12 months' treatment. Mean trough imatinib plasma levels were significantly higher in the group with complete cytogenetic response (56 patients) than in the group without (12 patients; P = .03) and higher in the group with major molecular response (MMR) than in the group without (34 patients [1452 +/- 649 ng/mL] versus 34 patients [869 +/- 427 ng/mL]; P < .001). Regarding trough imatinib plasma levels and their discrimination potential for MMR, the area under receiver operating characteristic curve was 0.775, with best sensitivity (77%) and specificity (71%) at a plasma threshold of 1002 ng/mL. Therefore, monitoring of imatinib plasma levels could be very useful for the management of patients with CML or should at least be checked in the case of treatment failure or suboptimal response.     

Loss of myeloid-related proteins 8 and myeloid-related proteins 14 expression in human esophageal squamous cell carcinoma correlates with poor differentiation

AIM: To study the expression of myeloid-related proteins(MRP)8 and myeloid-related proteins(MRP)14 in human esophageal squamous cell carcinoma and to investigate if there was any correlation between MRP8 and MRP14 expression level and histopathological grade in these tumors. METHODS: In this study, 65 cases of advanced esophageal squamous cell carcinoma were assessed for MRP8 and MRP14 expression using immunohistochemistry. Statistical analysis was performed for the comparison of MRP8 and MRP14 expression in normal and tumor tissues, and their relationship with clinicopathological features. RESULTS: Reduced or absent expression of MRP8 and MRP14 was observed in esophageal squamous cell carcinoma, with a significant difference between tumor tissues and normal tissues (P<0.01 and P<0.01 for MRP8 and MRP14, respectively). Poorly differentiated tumors presented a greater decrease than well and moderately differentiated tumors, with a correlation between their protein level and histopathological grading (P<0.001 and P<0.001, respectively). However, no significant association was found between MRP8 and MRP14 expression and age or gender (P>0.05). CONCLUSION: These findings suggest that the decreased expression of MRP8 and MRP14 might play an important role in the pathogenesis of human esophageal squamous cell carcinoma, being particularly associated with poor differentiation of tumor cells.     

Serum and mucosal S100 proteins, calprotectin (S100A8/S100A9) and S100A12, are elevated at diagnosis in children with inflammatory bowel disease

OBJECTIVE: Various markers characterize the complex inflammatory processes seen in chronic inflammatory bowel disease (IBD) including calprotectin, a complex of two S100 proteins, which has been evaluated and validated as a faecal marker of inflammation. However, the systemic and mucosal expression patterns of calprotectin and related S100 proteins are not well characterized in this disease. The objective of this study was to assess serum and mucosal levels of calprotectin, S100A12 and soluble receptor for advanced glycation end products (sRAGE), a putative S100 ligand, in a paediatric population with IBD. MATERIAL AND METHODS: Children were enrolled at diagnosis of IBD, along with groups of children without IBD. Standard inflammatory markers and disease activity scores were collated. Calprotectin, S100A12 and sRAGE levels in serum and biopsy culture supernatants were measured by ELISA and tissue distribution of S100 proteins was investigated by immunohistochemistry. RESULTS: Serum and mucosal calprotectin and S100A12 levels were increased in children with IBD as compared with non-IBD controls. Serum calprotectin levels correlated with S100A12 levels and with disease activity scores in children with IBD. sRAGE levels were not increased in IBD. S100A8, S100A9 and S100A12 were abundantly expressed throughout the lamina propria and epithelium in inflamed mucosa. In contrast, these proteins were present in the lamina propria, but not the epithelium, in non-inflamed mucosa. CONCLUSIONS: Serum calprotectin and S100A12 are increased in children with IBD and indicate disease activity. Elevated levels of these proteins are present in the colonic mucosa and may contribute to the pathogenesis of IBD. Furthermore, an imbalance between sRAGE and S100A12 may contribute to inflammatory changes present in IBD.     

S100 calgranulin proteins S100A8, S100A9 and S100A12 are expressed in the inflamed gastric mucosa of Helicobacter pylori-infected children.

The expression of the inflammatory S100 calgranulin proteins (S100A8, S100A9 and S100A12) in normal and Helicobacter pylori-infected gastric mucosa of children were examined. S100A8, S100A9 and S100A12, which were virtually absent in normal gastric mucosa, were highly expressed in H pylori-infected mucosa. This expression correlated with the severity of gastritis (r=0.9422, P<0.05). S100 calgranulins may be involved in bacterial-induced gastritis and may limit bacterial growth.     

Three-month treatment with pioglitazone reduces circulating levels of S100A8/A9 (MRP8/14) complex, a biomarker of inflammation, without changes in body mass index, in type 2 diabetics with abdominal obesity

We measured circulating S100A8/A9 (MRP8/14) complex levels before and after 3-month treatment with pioglitazone in type 2 diabetic patients. The results showed that pioglitazone reduced circulating S100A8/A9 complex levels, without changing body mass index, in type 2 diabetic patients with abdominal obesity.     

Calmodulin-binding proteins that interact with actin filaments in a Ca2+-dependent flip-flop manner: survey in brain and secretory tissues.

Regulatory actions of calmodulin on the contractile apparatus and cytoskeleton of smooth muscle and nonmuscle tissue are mediated by a number of specific calmodulin-binding proteins that bind to F-actin in a flip-flop manner--i.e., they bind to calmodulin or F-actin depending on the presence or absence, respectively, of Ca2+. A survey for such proteins in brain, adrenal gland, and pituitary gland identified six polypeptides on polyacrylamide gels--Mr 340,000 (band 1), Mr 240,000/235,000 doublet (band 2), Mr 150,000 (band 3), Mr 129,000 (band 4), Mr 105,000 (band 5), and Mr 94,000 (band 6)--as flip-flop-regulated calmodulin- and F-actin-binding polypeptides. In addition to these polypeptides, a Mr 58,000 non-flip-flop calmodulin-binding actin-binding polypeptide (band 7) was found in all tissues examined. Band 2 was identified as calspectin (spectrin-related protein; fodrin). The flip-flop regulation of calspectin required the presence of a heat-labile nondialyzable factor contained in a supernatant fraction of brain homogenates. Band 1 was distinct from microtubule-associated proteins (MAPs) 1 and 2. However, when band 1 polypeptide was kept on ice 3 days, it converted to a lower molecular weight doublet that migrated with MAP2 on NaDodSO4 gel electrophoresis. Bands 1 and 2 were found in all tissues examined.     

Cross-resistance to nalidixic acid, trimethoprim, and chloramphenicol associated with alterations in outer membrane proteins of Klebsiella, Enterobacter, and Serratia

We studied in vitro mutants of Klebsiella, Enterobacter, and Serratia cross-resistant to nalidixic acid, trimethoprim, and chloramphenicol that were similar to mutants found in vivo. The sole mechanism for this type of resistance appeared to be a reduction in permeability of the cell envelope. The mutants had significantly lower rates of uptake of glucose and chloramphenicol, but binding of chloramphenicol to ribosomes was normal. In addition, the amounts of dihydrofolate reductase were similar in both wild-type and cross-resistant mutants of Klebsiella. Examination of the bacterial outer membrane revealed that the amount of at least one major protein, with a molecular size of approximately 40 kilodaltons, was decreased in the mutants. Therefore the resistance seemed likely to be due to the reduction in quantity of these outer membrane proteins, possibly porins, in the mutant bacteria.     

DDX41 mutations in myeloid neoplasms are associated with male gender,TP53 mutations and high-risk disease

Myeloid neoplasms with germline DDX41 mutations have been incorporated into the 2017 WHO classification. Limited studies describing the clinicopathologic features and mutation profile are available. We searched for myeloid neoplasms with a DDX41 gene mutation tested by an 81-gene next-generation sequencing panel over a 7-month period. We identified 34 patients with myeloid neoplasms with DDX41 abnormalities; 26 (76%) men and 8 women (24%) [median age, 70 years], 20 acute myeloid leukemia (AML), 10 myelodysplastic syndrome (MDS), 1 chronic myelomonocytic leukemia (CMML) and 3 myeloproliferative neoplasms (MPN). Fifty-nine DDX41 variants were detected: 27 (46%) appeared somatic and 32 (54%) were presumably germline mutations. The majority of presumed germline mutations were upstream of the Helicase 2 domain (93%) and involved loss of the start codon (30%). The majority of somatic mutations were within the Helicase 2 domain (78%), with the missense mutation p.R525H being most common (67%). There was a significant difference in the location of germline or somatic mutations (P < .0001). Concomitant mutations were detected involving 19 genes, but only TP53 (n = 11, 32%), ASXL1 (n = 8, 24%), and JAK2 (n = 4, 12%) were recurrent. Twenty (59%) patients showed diploid cytogenetics. Twenty-three (68%) patients presented with AML or MDS-EB-2, suggesting an association with high-grade myeloid neoplasm. Patients with myeloid neoplasms carrying DDX41 mutations show male predominance (3:1), higher age at presentation, association with TP53 mutations, and association with high-grade myeloid neoplasms in our cohort at a referral cancer center setting. These findings support the recognition of myeloid neoplasms with DDX41 mutation as unique, need for germline confirmation, and further assessment of family members.     

CD14 and IL6 polymorphisms are associated with a pro-atherogenic profile in young adults with acute myocardial infarction

This study investigated the relationship of polymorphisms in genes encoding CD14, IL-6 and TLR4 with metabolic, inflammatory and endothelial markers in young adults with acute myocardial infarction (AMI). Glucose, lipids, nitrate and inflammatory markers, flow mediated vasodilatation (FMV) and flow mediated by nitrate (FMN) were evaluated in 102 AMI and 108 non-AMI (control group) young individuals (<45 years). CD14 ?260C>T (rs2569190), IL6 ?174G>C (rs1800795) and TLR4 c.896A>G (rs4986790) and TLR4 c.1196C>T (rs4986791) polymorphisms were analyzed by PCR–RFLP. Minor allele frequencies of CD14, IL6 and TLR4 polymorphisms were similar between AMI and control groups (p > 0.05). In AMI group, individuals carrying IL6 ?174CC genotype had higher serum triglycerides, VLDL cholesterol and glucose compared to the IL6 ?174GG/GC genotype carriers (p < 0.05). Multiple logistic analysis showed that IL6 ?174CC genotype carriers had increased risk for hyperglycemia (>5.77 mmol/l) [OR: 6.75, 95 % CI: 1.80–24.40, p = 0.004] and hypertriglyceridemia (>2.68 mmol/l) [OR: 3.00, 95 % CI: 1.00–9.00, p = 0.043]. Moreover, CD14 ?260TT genotype was associated with reduced serum HDL cholesterol [OR: 3.10, 95 % CI: 1.00–9.01, p = 0.044] and apolipoprotein AI [OR: 3.20, 95 % CI: 1.00–9.70, p = 0.038] in AMI group. Relationship between CD14 and IL6 variants and altered inflammatory and endothelial (nitrate, FMV and FMN) markers was not found in both AMI and control groups. The IL6 ?174G>C and CD14 ?260C>T polymorphisms are likely to be associated with a pro-atherogenic profile but not with increased inflammatory markers and endothelial dysfunction in young AMI patients.     

S100 proteins calprotectin and S100A12 are related to radiographic changes rather than disease activity in psoriatic arthritis with low disease activity

OBJECTIVE: To investigate serum levels of calprotectin (S100A8/S100A9) and S100A12 as markers of disease activity or distinct clinical or radiographic features in patients with psoriatic arthritis (PsA). METHODS: Serum levels of calprotectin and S100A12, erythrocyte sedimentation rate (ESR), and C-reactive protein (CRP) were determined in 119 patients with PsA. Correlations to clinical variables were calculated, and subgroups of patients were compared. RESULTS: The correlations to clinical disease activity measures were stronger for CRP than for ESR and calprotectin. In the regression analysis, calprotectin was identified as an independently associated factor for presence of peripheral radiographic features of arthritis (OR 1.33, 95% CI 1.01-1.76). S100A12 levels were also elevated in those with peripheral radiographic features (p = 0.036), but did not correlate with clinical variables of disease activity. CONCLUSION: Calprotectin and S100A12 do not perform better than traditional biomarkers of disease activity in PsA, but were associated with presence of peripheral radiographic features in this cross-sectional study. The patients' low level of disease activity may have led to underestimation of the associations between any biomarker and disease measures.