Adenoviral vector-mediated overexpression of IL-4 in the knee joint of mice with collagen-induced arthritis prevents cartilage destruction.
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Rheumatoid arthritis is a chronic inflammatory joint disease, leading to cartilage and bone destruction. In this study, we investigated the effects of local IL-4 application, introduced by a recombinant human type 5 adenovirus vector, in the knee joint of mice with collagen-induced arthritis. One intraarticular injection with an IL-4-expressing virus caused overexpression of IL-4 in the mouse knee joint. Enhanced onset and aggravation of the synovial inflammation were found in the IL-4 group. However, despite ongoing inflammation, histologic analysis showed impressive prevention of chondrocyte death and cartilage erosion. In line with this, chondrocyte proteoglycan synthesis was enhanced in the articular cartilage. This was quantified with ex vivo 35S-sulfate incorporation in patellar cartilage and confirmed by autoradiography on whole knee joint sections. Reduction of cartilage erosion was further substantiated by lack of expression of the stromelysin-dependent cartilage proteoglycan breakdown neoepitope VDIPEN in the Ad5E1 mIL-4-treated knee joint. Reduced metalloproteinase activity was also supported by markedly diminished mRNA expression of stromelysin-3 in the synovial tissue. Histologic analysis revealed marked reduction of polymorphonuclear cells in the synovial joint space in the IL-4-treated joints. This was confirmed by immunolocalization studies on knee joint sections using NIMP-R14 staining and diminished mRNA expression of macrophage-inflammatory protein-2 in the synovium tissue. mRNA levels of TNF-alpha and IL-1beta were suppressed as well, and IL-1beta and nitric oxide production by arthritic synovial tissue were strongly reduced. Our data show an impressive cartilage-protective effect of local IL-4 and underline the feasibility of local gene therapy with this cytokine in arthritis. (+info)
Notch1 expression in early lymphopoiesis influences B versus T lineage determination.
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Notch receptors regulate fate decisions in many cells. One outcome of Notch signaling is differentiation of bipotential precursors into one cell type versus another. To investigate consequences of Notch1 expression in hematolymphoid progenitors, mice were reconstituted with bone marrow (BM) transduced with retroviruses encoding a constitutively active form of Notch1. Although neither granulocyte or monocyte differentiation were appreciably affected, lymphopoiesis was dramatically altered. As early as 3 weeks following transplantation, mice receiving activated Notch1-transduced BM contained immature CD4+ CD8+ T cells in the BM and exhibited a simultaneous block in early B cell lymphopoiesis. These results suggest that Notch1 provides a key regulatory signal in determining T lymphoid versus B lymphoid lineage decisions, possibly by influencing lineage commitment from a common lymphoid progenitor cell. (+info)
Calcium-induced noncovalently linked tetramers of MRP8 and MRP14 detected by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry.
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MRP8 and MRP14 are members of the S100 family of calcium-binding proteins which play an important role during calcium-induced activation of phagocytes. Both proteins form noncovalently associated complexes as a prerequisite for biological functions. The exact stoichiometric composition of these complexes, however, has not been completely clarified yet. In the present study we show for the first time by ultraviolet matrix-assisted laser desorption/ionization mass spectrometry (UV-MALDI-MS) the calcium-induced formation of noncovalently associated (MRP8/MRP14)2 tetramers. Furthermore, we could determine posttranslational modifications of MRP8 and MRP14, the stoichiometric proportion of the two known MRP14 isoforms in the complexes as well as the number of calcium ions bound to the single MRP8 and MRP14 monomers and tetramers. MRP14 showed a higher affinity for calcium than MRP8. Upon complex formation the calcium binding increased to maximal saturation of the known EF hands in the complexed forms. Calcium-induced stabilization of the MRP8/MRP14 complexes was confirmed by DSC studies. Our results extend scope and application of UV-MALDI-MS by allowing identification of noncovalent protein complexes, the identification of minor alterations of subunits in such complexes as well as the determination of bound calcium ions. (+info)
Cell density-dependent growth in agar of bone marrow cells from tumor-bearing BALB/c mice in the absence of a colony-stimulating factor.
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Bone marrow cells from BALB/c mice with myeloid leukemia, lymphosarcoma, erythroblastosis, or mammary tumor produce small clusters in semisolid agar cultures in the absence of specific colony-stimulating factor. This spontaneous growth is observed only when high cell numbers (5 x 10 5 cells/ml) are plated. The phenomenon was encountered only when mice had an elevated number of mature or immature granulocytes in the peripheral blood. Removal of the adherent cells from the bone marrow did not abolish spontaneous growth, indicating that this colony-stimulating factor independency is not due to a high number of colony-stimulating cells in the bone marrow cells. This excluded the possibility that the spontaneous growth was due to a high endogenous stimulating activity of the bone marrow from tumor-bearing mice. (+info)
Detection of micrometastasis by cytokeratin 20 RT-PCR is limited due to stable background transcription in granulocytes.
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The reverse transcription polymerase chain reaction (RT-PCR) amplification of cytokeratin 20 (CK20) mRNA is considered a promising candidate method for the detection of circulating tumour cells in bone marrow and peripheral blood of cancer patients. In this study we have investigated the diagnostic specificity of the CK20 mRNA detection in samples from healthy donors (HD; n = 33), intensive care units patients (ICU; n = 20) and bone marrow obtained from patients suffering from chronic inflammatory diseases (CID; n = 14). RNAs purified from stabilized lysates showed positive results in 24% of the HD group (8/33), 35% of the ICU group (8/20) and in 40% of the CID group (5/14). The use of Ficoll gradients to separate nucleated cells completely restored the specificity of this CK20 RT-PCR assay. The CK20-expressing cells are positively identified to belong to the granulocyte fraction of leucocytes, which appear to express the gene on a background level. Our results demonstrate for the first time that CK20 mRNA expression is not limited to epithelium. Its occurrence in normal granulocytes has to be considered in tests designed to detect circulating cancer cells or micrometastases. (+info)
Molecular basis and enzymatic properties of glucose 6-phosphate dehydrogenase volendam, leading to chronic nonspherocytic anemia, granulocyte dysfunction, and increased susceptibility to infections.
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We have investigated the blood cells from a woman with a low degree of chronic nonspherocytic hemolytic anemia and frequent bacterial infections accompanied by icterus and anemia. The activity of glucose 6-phosphate dehydrogenase (G6PD) in her red blood cells (RBCs) was below detection level, and in her leukocytes less than 3% of normal. In cultured skin fibroblasts, G6PD activity was approximately 15% of normal, with 4- to 5-fold increased Michaelis constant (Km) for NADP and for glucose 6-phosphate. Activated neutrophils showed a decreased respiratory burst. Family studies showed normal G6PD activity in the RBCs from all family members, including both parents and the 2 daughters of the patient. Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA showed a novel, heterozygous 514C-->T mutation, predicting a Pro172-->Ser replacement. Analysis of G6PD RNA from the patient's leukocytes and fibroblasts showed only transcripts with the 514C-->T mutation. This was explained by the pattern of X-chromosome inactivation, studied by means of the human androgen receptor (HUMARA) assay, which proved to be skewed in the patient, her mother, and one of the patient's daughters. Thus, the patient has inherited a de novo mutation in G6PD from her father and an X-chromosome inactivation determinant from her mother, causing exclusive expression of the mutated G6PD allele. Purified mutant protein from an Escherichia coli expression system showed strongly decreased specific activity, increased Km for NADP and for glucose 6-phosphate, and increased heat lability, which indicates that the defective phenotype is due to 2 synergistic molecular dysfunctions: decreased catalytic efficiency and protein instability. (+info)
Mussel defensins are synthesised and processed in granulocytes then released into the plasma after bacterial challenge.
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MGD1 (Mytilus galloprovincialis defensin 1), a new member of the arthropod defensin family, is a 4 kDa antibacterial peptide previously isolated from the plasma of Mediterranean mussels. We report here the presence of MGD1 in the organelle-rich fraction of hemocytes and the cDNA sequence corresponding to MGD1 and one new isoform mRNA: MGD2. Sequence analysis indicated that MGDs are synthesised as precursors consisting of a putative signal peptide of 21 residues, the active peptide of 39 amino acids and a 21 residue carboxyl-terminal extension, rich in acidic amino acids. Localisation of the transcripts by northern blot revealed that the precursors are abundantly expressed in hemocytes. Immunocytochemistry at both the optical and ultrastructural levels showed that defensins (i) are predominantly located in vesicles of a granulocyte subclass of hemocytes containing small granules, (ii) are also found in large clear granules of another granulocyte subclass, and (iii) that MGD immune reactivity existed in granular structures of enterocytes. Finally, we revealed that bacterial challenge triggered a plasmatic increase of MGD1 concentration and gave evidence of the simultaneous release of the peptides from the hemocytes. (+info)
Acute leukemia in adults: assessment of remission induction with combination chemotherapy by clinical and cell-culture criteria.
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Remission induction was assessed by clinical and cell-culture criteria for 65 patients with acute myelogenous leukemia (AML), 11 patients with chronic myelogenous leukemia (CML) in blast crisis and 19 patients with acute lymphoblastic leukemia (ALL). Cyclophosphamide, cytosine arabinoside and vincristine (CAV) therapy resulted in complete remission in 23 of 50 previously untreated patients with AML and in 3 of the 11 patients with CML. Fourteen patients with ALL responded to vincristine-prednisone induction therapy and two to induction therapy with CAV. The median duration of survival of the responding patients was 2.2 years, compared with 4 months for the patients who did not respond to treatment. Granulopoietic colony formation, assessed by assay of colony-forming units dependent on colony-stimulating activity in culture (CFU-C), was abnormal in 37 of 42 bone marrow aspirates from patients with AML before treatement. CFU-C concentration increased when leukocyte-conditioned medium (LCM) was added to the cultures; 13 cultures had normal or elevated CFU-C concentration with LCM. Marrow cells of patients with ALL or CML in blast crisis demonstrated a similar pattern. Serial studies of marrow CFU-C concentration of 31 patients with AML demonstrated a change to a normal pattern with successful remission induction. Results of this study suggest that administration of purified LCM to leukemic patients might increase granulocyte production from potential but unstimulated granulopoietic precursors. This therapy would lessen the probability of death from infection during remission induction. (+info)