Length of uninterrupted CGG repeats determines instability in the FMR1 gene.

Analysis of 84 human X chromosomes for the presence of interrupting AGG trinucleotides within the CGG repeat tract of the FMR1 gene revealed that most alleles possess two interspersed AGGs and that the longest tract of uninterrupted CGG repeats is usually found at the 3' end. Variation in the length of the repeat appears polar. Alleles containing between 34 and 55 repeats, with documented unstable transmissions, were shown to have lost one or both AGG interruptions. These comparisons define an instability threshold of 34-38 uninterrupted CGG repeats. Analysis of premutation alleles in Fragile X syndrome carriers reveals that 70% of these alleles contain a single AGG interruption. These data suggest that the loss of an AGG is an important mutational event in the generation of unstable alleles predisposed to the Fragile X syndrome.

Protective effects of C5a blockade in sepsis.

Sepsis in humans is a difficult condition to treat and is often associated with a high mortality rate. In this study, we induced sepsis in rats using cecal ligation and puncture (CLP). In rats depleted of the complement factor C3, CLP led to very short survival times (about 4 days). Of the rats that underwent CLP ('CLP rats') that were C3-intact and treated with preimmune IgG, most (92%) were dead by 7 days. Blood neutrophils from these rats contained on their surfaces the powerful complement activation product C5a. This group had high levels of bacteremia, and their blood neutrophils when stimulated in vitro had greatly reduced production of H2O2, which is known to be essential for the bactericidal function of neutrophils. In contrast, when companion CLP rats were treated with IgG antibody against C5a, survival rates were significantly improved, levels of bacteremia were considerably reduced, and the H2O2 response of blood neutrophils was preserved. Bacterial colony-forming units in spleen and liver were very high in CLP rats treated with preimmune IgG and very low in CLP rats treated with IgG antibody against C5a, similar to values obtained in rats that underwent 'sham' operations (without CLP). These data indicate that sepsis causes an excessive production of C5a, which compromises the bactericidal function of neutrophils. Thus, C5a may be a useful target for the treatment of sepsis.

The chemosuppression of chemotaxis.

The effects of various drugs on chemotaxis of polymorphonuclear leukocytes (PMN's) in vitro and in vivo have been studied. Response of rabbit PMN's in vitro to the chemotactic factor of rabbit serum, consisting of an activated protein-protein complex of the fifth and sixth (and probably seventh) components of complement (C'), is suppressed by hydrocortisone, methyl prednisolone, and chloroquine. Drug concentrations causing 50% inhibition of chemotaxis in vitro were found to be: hydrocortisone, 2.9 x 10(-4)M; methly prednisolone, 1.2 x 10(-4)M; and chloroquine 8.5 x 10(-6)M. The hydrocortisone effect on PMN's appeared to be irreversible, since washing of the cells did not restore their chemotactic response. 2, 4-Dinitrophenol (DNP), vitamin A, and endotoxin did not inhibit chemotaxis. Hydrocortisone and chloroquine did block serum C' activity in vitro, but only at substantially higher concentrations. Using the reversed passive Arthus reaction in guinea pigs as a model for chemotaxis in vivo, systemic treatment of animals with hydrocortisone or chloroquine inhibited development of the vasculitis. Circulating antigen and C' were fixed in vascular structures, and serum C' was not perceptibly altered. Nevertheless, PMN infiltrates failed to occur. Local administration of hydrocortisone also prevented influx of PMN's in the Arthus reaction, in spite of the fact that immune reactants were found fixed in the vascular walls. Systemic treatment of guinea pigs with DNP did not diminish the intensity of the Arthus reactions. Phagocytosis of zymosan particles by rabbit PMN's was inhibited by hydrocortisone, methyl prednisolone, and chloroquine, but not by DNP or endotoxin. The concentrations of drugs inhibitory in phagocytosis were substantially higher than those required for inhibition of chemotaxis in vitro. These findings suggest that hydrocortisone and chloroquine inhibit the inflammatory process by preventing the response of leukocytes to chemotactic stimuli.

A plasmin-split fragment of C'3 as a new chemotactic factor.

When streptokinase and highly purified human plasminogen are added to human serum or to partly purified or highly purified preparations containing the third component of complement (C'3), either rabbit or human, a chemotactic factor is generated. This chemotactic factor is a split product of C'3 and is dialyzable, fast moving electrophoretically, slowly sedimenting in sucrose density gradient ultracentrifugation, and has an approximate molecular weight of 6000. It is calculated that this fragment accounts for approximately 4% of the intact molecule. The C'3 fragment has the following biologic properties: It is chemotactic for rabbit PMN's in vitro, it causes accumulation of PMN's in vivo, and it increases vascular permeability in rat skin. In addition to generating a chemotactic factor, plasmin destroys the complement-associated chemotactic factor that is a trimolecular complex consisting of the fifth (C'5), sixth (C'6), and seventh (C'7) components of complement. This has been shown by a loss of chemotactic activity, as well as a dissociation of the C'5, C'6, C'7 complex and a destruction of C'6 hemolytic activity. The biologic significance of the plasmin-generated chemotactic factor is discussed in relation to other recently discovered biologically active fragments of C'3.

Chemotoxis of mononuclear cells.

Chemotaxis of rabbit mononuclear cells was studied by the micropore-filter technique. Mononuclear cells obtained from mineral oil-induced peritoneal exudates respond chemotactically to rabbit serum treated with immune complexes or with streptokinase and plasminogen, to soluble factors produced by bacteria, and to lysates obtained from rabbit neutrophils. The first chemotactic factor requires heat-labile factors in serum for its generation, but, once formed, the chemotactic factor is relatively heat stable. This factor has been compared with the complement-associated factor in serum, C'(5, 6, 7)a, that is chemotactic for neutrophils. The ability to generate the mononuclear cell chemotactic factor in serum that has been treated with potassium thiocyanate suggests that complement is not required. The position of the chemotactic factor in preparative electrophoresis and density-gradient ultracentrifugation indicates that on the basis of physical-chemical criteria, this factor is not C'(5, 6, 7)a. The mononuclear cell chemotactic factor present in lysates of neutrophils sediments slowly in the ultracentrifuge and may be related, at least in part, to cationic peptides of lysosomal granules. A study in the time course of the chemotactic response of mononuclear cells reveals that the response begins to level off after 4 or 5 hr. This is in sharp contrast to the time course for the chemotactic response of neutrophils, in which the reaction is complete within 1.5 hr. Rabbit mononuclear cells obtained from a starch-induced peritoneal exudate respond to serum treated with plasminogen and streptokinase and to the soluble factor produced by bacteria, but no chemotactic response is elicited to serum treated with immune complexes. This indicates a functional difference between two populations of mononuclear cells. Rabbit alveolar macrophages respond poorly to all agents tested, although a weak chemotactic response to bacterial factors was found. The requirement of a serine esterase in the mononuclear cell for the cell to respond chemotactically was defined by the use of organophosphorus inhibitors. Pretreatment of mononuclear cells with several series of phosphonates renders them unresponsive in the chemotactic system. The effect is similar for mononuclear cells responding chemotactically to activated serum and to the bacterial chemotactic factor. Several points of contrast with inhibition profiles obtained in the chemotactic system of the neutrophil suggest that, while the mononuclear cell requires a serine esterase for chemotactic responsiveness, this enzyme is different from the one previously defined in the neutrophil.

Complement-derived leukotactic factors in pathological fluids.

Leukotactic factors derived from the complement sequence consist of C5-related products, including the small cleavage product and the higher molecular weight complex (C567). A leukotactically active cleavage product (of low molecular weight) has also been described. The C5-related factors have been found in two conditions: soluble tissue extracts of immunologic vasculitis and in synovial fluids from patients with rheumatoid arthritis. The C3-related product has been found in synovial fluids from patients with inflammatory, nonrheumatoid arthritis and in extracts of experimentally infarcted myocardium. The presence of these factors may be causally related to the acute inflammatory nature of the cellular exudates.