Purification of crystal induced chemotactic factor from human neutrophils.

The crystal induced chemotactic factor is a protein that is released by neutrophils in response to a phagocytic stimulus like monosodium urate or calcium pyrophosphate crystals. This protein has been postulated to play an important role in the development of crystal-induced arthritis. In this report, we present a protocol for its purification which involved ammonium sulfate fractionation, affinity chromatography, lectin chromatography, and high pressure chromatography (HPLC). The purified protein migrated as a single band on SDS-PAGE. Based on its electrophoretic and chromatographic (HPLC) properties, it appears to have a m.w. of 15,000. It has a blocked N-terminal since our attempt to sequence the chemotactic protein by established procedures failed. The protein contains one methionine residue which appears to be essential for its chemotactic activity since cyanogen bromide treatment abolished its chemotactic activity. To our knowledge this is the first report on purification of this autocrine leukoattractant from human neutrophils.

The natural course of central retinal vein occlusion.

We reviewed the records of 160 patients who had central retinal vein occlusion between 1980 and 1985. Of 168 eyes, 107 (64%) were classified as nonischemic types and 61 (36%) were classified as ischemic types. Of 107 nonischemic eyes, ten (9%) converted to the ischemic variant. Of 107 nonischemic eyes, 33 (31%) lost three or more lines of visual acuity irrespective of initial visual acuity. A final visual acuity less than or equal to 20/200 was recorded in 57 of 61 (93%) of ischemic eyes and 53 of 107 (50%) of nonischemic eyes.

Monoclonal antibody to the crystal induced chemotactic factor from human neutrophils detects similar immunoreactive and biologically active protein in promyelocytic HL-60 cells.

The crystal induced chemotactic factor (CCF) is a protein released by human neutrophils (PMN-CCF) that phagocytize monosodium urate or calcium pyrophosphate crystals. This protein has been thought to play an important role in crystal induced arthritis. In the present report, a mouse hybridoma cell line A2900C2.12 which specifically secretes monoclonal antibody (MAb) to CCF has been developed. The antibody secreted by the cell line is mostly IgG2b and positive for both the light chains kappa and lambda. The MAb has been purified by affinity chromatography using a protein-A column. The purified MAb was found to be over 95% pure based on the results from SDS-PAGE. The MAb has been used to demonstrate that a leukotactic protein is released from polymorphonuclear leukocytes (PMN) and promyelocytic HL-60 cells in response to a phagocytic or degranulating stimuli such as monosodium urate crystals or N-formyl-Met-Leu-Phe (FMLP), respectively. "Western blots" and silver nitrate staining of the purified leukotactic protein from PMNs and HL-60 cells show that these proteins have the same immunodeterminant site(s) and both appear to have a molecular weight (m.w.) of about 15,000. A simple purification protocol including immunoaffinity high pressure liquid chromatography (HPLC) has been described for purifying chemotactic proteins with the same antigenic determinant as the PMN-CCF.

Abnormal lipoprotein in uraemic patients treated conservatively and by maintenance haemodialysis.

Observations of serum lipids and lipoproteins in 35 uraemic patients treated conservatively (20 cases) and by maintenance haemodialysis (15 cases) are presented. Serum triglycerides levels of both groups (243.7 +/- 119.1 and 160.8 +/- 55.0 mg/dl) were significantly higher (p less than 0.001) and high density lipoprotein cholesterol (HDLC) levels (19.0 +/- 6.1 and 12.17 +/- 5.1 mg/dl) were significantly lower (p less than 0.001) in comparison to those in 20 controls. Agarose gel electrophoresis revealed a second, slow migrating pre-beta band in significantly higher percentage of uraemics (55% and 93.3%) as compared to controls (25%). Until the effects of uraemia and chronic haemodialysis on lipoprotein metabolism are better understood, caution should be exercised in the interpretation of HDLC levels in these patients.

Effect of tunicamycin C2 on the induction of crystal induced chemotactic factor in human neutrophils.

We have investigated the effect of tunicamycin-C2, which specifically inhibits glycosylation of proteins but not their synthesis, in the induction of crystal-induced chemotactic factor (CCF) in human neutrophils by monosodium urate crystals. Tunicamycin C2 appears to impair transport of the CCF to the lysosomal fraction of the cell and its subsequent secretion into the extracellular fluid, resulting in accumulation of nonglycosylated chemotactic proteins in the cytosol. Based on the results of affinity chromatography studies with various lectins, the carbohydrate moiety(ies) of the glycosylated chemotactic peptide contain mannose, N-acetyl-D-galactosamine, galactose, and fucose.

The risk for systemic vascular diseases and mortality in patients with central retinal vein occlusion.

In this cross-sectional study, the authors evaluated 197 patients diagnosed with central retinal vein occlusion (CRVO) at the Wilmer Ophthalmological Institute between 1980 and 1985 to determine the risk of systemic disease and mortality. Complete follow-up information for mortality was obtained in 191 (97%). National Health Interview Survey (NHIS) patients and Wilmer cataract patients formed two comparison groups. The prevalence of hypertension was significantly elevated in the CRVO cases when compared with both comparison groups (P less than 0.03, 0.005). The prevalence of diabetes mellitus was increased in CRVO cases in comparison with the NHIS group (P less than 0.005). The prevalence of cerebrovascular or cardiovascular disease was the same for all three groups, as was overall mortality. Mortality was not increased in CRVO cases as compared with United States mortality rates.

Reduced cell migration and disruption of the actin cytoskeleton in calpain-deficient embryonic fibroblasts.

The physiological functions and substrates of the calcium-dependent protease calpain remain only partly understood. The mu- and m-calpains consist of a mu- or m-80-kDa large subunit (genes Capn1 and Capn2), and a common 28-kDa small subunit (Capn4). To assess the role of calpain in migration, we used fibroblasts obtained from Capn4(-/-) mouse embryos. The cells lacked calpain activity on casein zymography and did not generate the characteristic calpain-generated spectrin breakdown product that is observed in wild-type cells. Capn4(-/-) cells had decreased migration rates and abnormal organization of the actin cytoskeleton with a loss of central stress fibers. Interestingly, these cells extended numerous thin projections and displayed delayed retraction of membrane protrusions and filopodia. The number of focal adhesions was decreased in Capn4(-/-) cells, but the cells had prominent vinculin-containing focal complexes at the cell periphery. The levels of the focal adhesion proteins, alpha-actinin, focal adhesion kinase (FAK), spectrin, talin, and vinculin, were the same in Capn4(+/+) and Capn4(-/-) cells. FAK, alpha-actinin, and vinculin were not cleaved in either cell type plated on fibronectin. However, proteolysis of the focal complex component, talin, was detected in the wild-type cells but not in the Capn4(-/-) cells, suggesting that calpain cleavage of talin is important during cell migration. Moreover, talin cleavage was again observed when calpain activity was partially restored in Capn4(-/-) embryonic fibroblasts by stable transfection with a vector expressing the rat 28-kDa calpain small subunit. The results demonstrate unequivocally that calpain is a critical regulator of cell migration and of the organization of the actin cytoskeleton and focal adhesions.