Secretion by human fibroblasts of monocyte chemoattractant protein-1, the product of gene JE

We recently purified human monocyte chemoattractant protein-1 (MCP-1) from culture fluids of either human glioma cell lines or mitogen-stimulated human peripheral blood mononuclear leukocytes. It has now been shown that MCP-1 is the product of the gene JE, which was first recognized by its expression in fibroblasts stimulated with platelet-derived growth factor (PDGF). We therefore studied secretion of MCP-1 by three human fibroblast cell lines. Monocyte chemotactic activity was found in culture fluids of all three lines after growth to confluence in DMEM-10% FCS, and the amounts secreted per cell were comparable for the three lines. The MRC-5 line was chosen for further study. Monocyte chemotactic activity secretion by confluent MRC-5 cultures continued after a switch to serum-free medium and was not inhibited by anti-PDGF antibody, indicating that secretion may not have been caused by autocrine release of PDGF. When concentrated serum-free MRC-5 culture fluid was injected into an HPLC gel filtration column, only one chemotactic activity peak was observed, which was in the same location as glioma-derived MCP-1. The activity was completely absorbed out by an anti-MCP-1 affinity column, which indicates that all the chemotactic activity in MRC-5 culture fluid was accounted for by MCP-1. PDGF caused a marked increase in chemotactic activity over that found in serum-free culture fluid of MRC-5 or 501T cells. Immunoprecipitation by anti-human MCP-1 showed two bands, corresponding to the two forms of MCP-1 previously described (MCP-1 alpha and beta); and the amounts increased in response to PDGF stimulation. Thus, the reported increase in human fibroblast JE mRNA in response to PDGF-containing serum stimulation is reflected in increased secretion of the MCP-1 gene product.     

Identification of high affinity receptors for human monocyte chemoattractant protein-1 on human monocytes

The binding of human monocyte chemoattractant protein-1 (MCP-1) to human monocytes was studied. MCP-1 was radioiodinated with Iodo-beads (Pierce Chemical Co., Rockford, IL) without significant loss of biologic activity. 125I-MCP-1 binding to PBMC occurred within 5 min at 0 degrees C and the binding was inhibited by unlabeled MCP-1 dose dependently but not by neutrophil attractant/activation protein-1 or FMLP. 125I-MCP-1 bound to monocytes; no significant binding to either neutrophils or lymphocytes was observed. Scatchard plot analysis indicated that monocytes had a minimum of 1700 +/- 600 binding sites per cell with a Kd of 1.9 +/- 0.2 x 10(-9) M. For analysis of binding by flow cytometry, MCP-1 was biotinylated. In contrast to radioiodination, biotinylation resulted in loss of activity; potency was 10-fold less, but the efficacy was retained. Detection by flow cytometry of bound biotinylated MCP-1 with avidin-FITC confirmed results obtained with 125I-MCP-1. Biotinylated MCP-1 bound to monocytes but not to lymphocytes; and the binding was inhibited by a 100-fold excess of unlabeled MCP-1.     

Leukocyte specificity and binding of human neutrophil attractant/activation protein-1

Neutrophil attractant/activation protein-1 (NAP-1) was previously shown to attract human neutrophils, but not monocytes. The purpose of this study was to determine if NAP-1 interacted with other types of blood leukocytes. In addition to its chemotactic activity for neutrophils, NAP-1 induced chemotactic responses by T lymphocytes and basophils. Chemotactic potency (10(-8) M for an optimal response) was the same for all three cell types. However, NAP-1 caused a chemotactic response in excess of random migration of 7% or 16% of basophils (depending on the medium used) and only 9% of T lymphocytes, in contrast to 30% of neutrophils. This agonist was not chemotactic for partially purified normal human eosinophils. The symmetrical histogram obtained by flow cytometry of neutrophils equilibrated at 0 degree C with fluoresceinated NAP-1 indicates that all neutrophils bound the ligand. A dose-response curve plateau, and inhibition of binding of NAP-1-FITC by unlabeled ligand are evidence for saturable binding to receptors, estimated to be 7000 per cell. Our results suggest that, for induction of an acute inflammatory response, the quantitatively significant action of NAP-1 is on neutrophils.     

A New Enzymatic Cycling Technique for Glutamate Determination in Brain Microdialysates

A quantitative analysis of glutamate in brain dialysate was made by using an enzymatic cycling technique. This method made it possible to measure the concentration of glutamate in dialysate collected at 30-s intervals. Dialysates were collected from Mongolian gerbil hippocampus before, during, and after two 90-s ischemic insults at an interval of 5 min. An extracellular increase in levels of glutamate was already observed in samples collected during a 30-60 s period after the onset of each ischemia, and the levels of glutamate were maximal at the end of each period of ischemia (approximately a fourfold increase). The increased levels of glutamate rapidly returned almost to preischemic levels by 30 s of recirculation. This method will provide more precise information about temporal changes in the extracellular glutamate concentration in the brain during ischemia.     

Production and characterization of recombinant human neutrophil chemotactic factor

A putative mature human neutrophil chemotactic factor (NCF) corresponding to the C-terminal 72 amino acids of its precursor was directly produced in Escherichia coli by recombinant DNA technology. Human NCF was present in both the soluble and insoluble protein fractions of the homogenate of host cells, and it was partially purified as a water-soluble polypeptide from both fractions, separately. The partially purified NCF preparation was highly purified to an endotoxin-free homogeneous polypeptide by means of CM-Sepharose CL-6B column chromatography and gel filtration on Toyopearl HW-55. No difference between the human NCF preparations purified from both starting materials could be found concerning purity, primary structure, solubility, molecular weight, and chemotactic activity for human neutrophils. The amino acid sequence of recombinant human NCF was identical to the sequence deduced from the cDNA sequence. A methionine residue due to the translation initiation codon was removed. Recombinant human NCF was found to be biologically active and to exhibit chemotactic activity for human neutrophils in vitro and cause a neutrophil infiltration in vivo in mice.     

The vasculature of the peroneal tissue transfer

Peroneal vascularized composite-tissue transfer has many useful applications and advantages. An anatomic study of the peroneal artery and vein and their branches was carried out on 80 adult cadaver legs. The number of cutaneous branches averaged 4.8 +/- 1.4 per leg. The length of the cutaneous branches averaged 5.4 +/- 1.5 cm. The external diameters of cutaneous branches at the skin distribution site were 0.6 +/- 0.2 mm for the artery and 0.8 +/- 0.3 mm for the vein. The communicating branches were branched at anterior or posterior tibial vessels 6.1 +/- 2.4 cm proximal to the lateral malleolus. The range of rotation of the island flap when transposed proximally was 14.3 +/- 3.3 cm proximal from the head of the fibula, and when transposed distally, the range of rotation was 16.9 +/- 5.3 cm distally.     

Tracheoplasty with palatal mucoperiosteal graft

A case of tracheal reconstruction with a palatal mucoperiosteal graft is reported. The patient is a 75-year-old woman who had the anterior wall of her trachea resected for invading thyroid carcinoma. Palatal mucoperiosteum, which is easy to handle, provided both the lining and supporting tissue for the trachea. Local neck skin was used for covering. This procedure proved very successful.     

Augmentation of the nostril splint for retaining the corrected contour of the cleft lip nose

Whatever method is used to correct the deformity of the cleft lip nose, it is important to maintain the corrected contour of the nose for a certain period postoperatively. For this purpose, moldable silicone rubber is used to add volume to a ready-made nostril splint. With this material it is easy to make a splint that fits the individual contour of the nostril. Clinical examples are presented.