Cytokine production in a model of wound healing: the appearance of MIP-1, MIP-2, cachectin/TNF and IL-1

Macrophages are essential for normal wound repair and many of their effects on healing wounds are likely to be mediated by the secretion of cytokines. This study examines the appearance of messenger RNA (mRNA) for cachectin/tumor necrosis factor (TNF), IL 1, and macrophage inflammatory proteins 1 and 2 (MIP-1 and MIP-2), as well as the mature peptides, in a model of wound healing using wound chambers. RNA for all four cytokines can be detected in wound inflammatory cells by polymerase chain reaction amplification throughout the first 7 days. Cachectin/TNF and IL 1 protein levels peaked on the first day after wound chamber implantation, and MIP-1 and MIP-2 were detected only on day 3. The data suggest that these cytokines participate in the early inflammatory response to wounding.     

PEG-mediated expression of GUS and CAT genes in protoplasts from embryogenic suspension cultures of Picea glauca

ß-Glucuronidase (GUS) and chloramphenicol acetyl transferase (CAT) were used as reporter proteins in protoplasts from embryogenic suspension cultures of Picea glauca (Moench) Voss (white spruce). Plasmid DNA enclosing chimeric GUS and CAT constructs, using the cauliflower mosaic virus 35S promoter, was introduced into Picea glauca protoplasts using polyethylene glycol (PEG). Transient expression was detected 12 to 40 h after PEG-mediated DNA delivery. Dose-response curves using covalently closed circular plasmid DNA, in the absence of carrier DNA, have been obtained for each of these reporter genes. Linearized plasmid DNA gave lower levels of expression than covalently closed circular plasmid DNA when assayed 40 h after PEG-mediated DNA transfer. The use of carrier DNA (herring sperm DNA), in combination with covalently closed circular plasmid DNA, increased the level of expression of GUS by about 50%. CAT expression was enhanced if PEG-mediated delivery was performed on ice rather than at room temperature. The highest level of expression for CAT, and the lowest signal-to-noise ratio, was found 24 h after PEG-mediated DNA transfer. Both GUS and CAT provided results that were quantifiable and can therefore be used as reporter genes in Picea glauca.Abbreviations CAT chloramphenicol acetyl transferase - GUS ß-glucuronidase - CaMV cauliflower mosaic virus - NOS nopaline synthase - CCC covalently closed circular DNA - L linear DNA - PEG polyethylene glycol - HS herring sperm DNA - P protoplasts - PCM protoplast culture medium - MES morpholinoethane-sulfonic acid - Cm chloramphenicol - Ac acetylated - MUG 4-methyl umbelliferyl ß-D-glucuronide - TLC thin layer chromatography     

Derivation and characterization of an efficiently myocarditic reovirus variant.

A reovirus variant, 8B, was isolated from a neonatal mouse which had been inoculated with a mixture of two reovirus strains: type 1 Lang (T1L) and type 3 Dearing (T3D) (E. A. Wenske, S.J. Chanock, L. Krata, and B. N. Fields, J. Virol. 56:613-616, 1985). 8B is a reassortant containing eight gene segments derived from the T1L parent and two gene segments derived from the T3D parent. Upon infection of neonatal mice, 8B produced a generalized infection characteristic of many reoviruses, but it also efficiently induced numerous macroscopic external cardiac lesions, unlike either of its parents. Microscopic examination of hearts from infected mice revealed myocarditis with necrotic myocytes and both polymorphonuclear and mononuclear cellular infiltration. Electron microscopy revealed viral arrays in necrotic myocytes and dystrophic calcification accompanying late lesions. Determination of viral titers in hearts from T1L-, T3D-, or 8B-infected mice indicated that growth was not the primary determinant of myocardial necrosis. Results from inoculations of athymic mice demonstrated that T cells were not a requirement for the 8B-induced myocarditis. Finally, 8B was more cytopathic than either of the parent viruses in cultured mouse L cells. Together, the data suggest that 8B-induced myocardial necrosis is due to a direct effect of reovirus on myocytes. Reovirus thus provides a useful model for the study of viral myocarditis.     

Lymphocytes protect against and are not required for reovirus-induced myocarditis.

Many studies suggest that host lymphocytes are damaging, rather than protective, in virally induced myocarditis. We have investigated the role of lymphocyte-based immunity in murine myocarditis by using a myocarditic reovirus (reovirus serotype 3 8B), nonmyocarditic reoviruses, adoptive transfer experiments, and mice with severe combined immunodeficiency (SCID mice). Prior to infection, passive transfer of monoclonal antibodies specific for 8B capsid proteins protected neonatal mice against 8B-induced myocarditis, indicating that humoral immunity can protect against myocarditis. Some monoclonal antibodies acted by blocking viral spread to and/or replication in the heart. Passive transfer of reovirus-immune, but not naive, spleen cells prior to infection protected neonatal mice from 8B-induced myocarditis. Depletion of either CD4 or CD8 T cells resulted in increased viral titer in the heart but did not abrogate immune cell-mediated protection against myocardial injury. This shows that both CD4 and CD8 T cells can act independently to protect myocardial tissue from reovirus infection. In addition, reovirus 8B caused extensive myocarditis in SCID mice. This confirms a prior report (B. Sherry, F. J. Schoen, E. Wenske, and B. N. Fields, J. Virol. 63:4840-4849, 1989) that T cells are not required for reovirus-induced myocarditis and demonstrates for the first time that B cells are not required for reovirus-induced myocarditis. We used SCID mice and a panel of reoviruses to assess (i) the relationship between growth in the heart and myocardial damage and (ii) the possibility that nonmyocarditic reoviruses exhibit a myocarditic phenotype in the absence of functional lymphocytes. Growth in the heart was not the sole determinant of myocarditic potential in SCID mice. Although 8B induced myocarditis in SCID mice, no or minimal myocarditis was found in SCID mice infected with four reovirus strains previously shown (B. Sherry and B. N. Fields, J. Virol. 63:4850-4856, 1989) to be nonmyocarditic or poorly myocarditic in normal neonatal mice. We conclude that (i) humoral immunity and cellular immunity are protective against, and not required for, reovirus-induced myocarditis and (ii) the potential to induce cardiac damage is a property of the virus independent of lymphocyte-based immunity.     

Macrophage inflammatory protein (MIP)-1 beta abrogates the capacity of MIP-1 alpha to suppress myeloid progenitor cell growth

The effects of recombinant murine macrophage inflammatory protein (MIP)-1 beta and MIP-2 on the suppressive activity of MIP-1 alpha were tested using colony formation by human and murine bone marrow burst-forming unit-erythroid (BFU-E), colony-forming unit-granulocyte erythroid macrophage, megakaryocyte (CFU-GEMM), and colony-forming unit-granulocyte macrophage (CFU-GM) progenitor cells. MIP-1 beta, but not MIP-2, when added with MIP-1 alpha to cells, blocked the suppressive effects of MIP-1 alpha on both human and murine BFU-E, CFU-GEMM, and CFU-GM colony formation. Similar results were observed regardless of the early acting cytokines used: human rGM-CSF plus human rIL-3, and two recently described potent cytokines, a genetically engineered human rGM-CSF/IL-3 fusion protein and MGF, a c-kit ligand. The more potent the stimuli, the greater the suppressive activity noted. Pulse treatment of hu bone marrow cells with MIP-1 alpha at 4 degrees C for 1 h was as effective in inhibiting colony formation as continuous exposure of cells to MIP-1 alpha, and the pulsing effect with MIP-1 alpha could not be overcome by subsequent exposure of cells to MIP-1 beta. Also, pulse exposure of cells to MIP-1 beta blocked the activity of subsequently added MIP-1 alpha. For specificity, the action of a nonrelated myelosuppressive factor H-ferritin, was compared. MIP-1 alpha and H-ferritin were shown to act on similar target populations of early BFU-E, CFU-GEMM, and CFU-GM. MIP-1 beta did not block the suppressive activity of H-ferritin. Also, hemin and an inactive recombinant human H-ferritin mutein counteracted the suppressive effects of the wildtype H-ferritin molecule, but did not block the suppressive effects of MIP-1 alpha. These results show that MIP-1 beta's ability to block the action of MIP-1 alpha is specific. In addition, the results suggest that MIP-1 alpha and MIP-beta can, through rapid action, modulate early myeloid progenitor cell proliferation.     

The structure of human mitochondrial DNA variation

Summary Restriction analysis of mitochondrial DNA (mtDNA) of 3065 humans from 62 geographic samples identified 149 haplotypes and 81 polymorphic sites. These data were used to test several aspects of the evolutionary past of the human species. A dendrogram depicting the genetic relatedness of all haplotypes shows that the native African populations have the greatest diversity and, consistent with evidence from a variety of sources, suggests an African origin for our species. The data also indicate that two individuals drawn, at random from the entire sample will differ at approximately 0.4% of their mtDNA nucleotide sites, which is somewhat higher than previous estimates. Human mtDNA also exhibits more interpopulation heterogeneity (G_ST=0.351±0.025) than does nuclear DNA (G_ST=0.12). Moreover, the virtual absence of intermediate levels of linkage disequilibrium between pairs of sites is consistent with the absence of genetic recombination and places constraints on the rate of mutation. Tests of the selective neutrality of mtDNA variation, including the Ewens-Watterson and Tajima tests, indicate a departure in the direction consistent with purifying selection, but this departure is more likely due to the rapid growth of the human population and the geographic heterogeneity of the variation. The lack of a good fit to neutrality poses problems for the estimation of times of coalescence from human mtDNA data.     

Acetylcholine receptor clusters are associated with nuclei in rat myotubes

Clustered and diffuse acetylcholine receptors are present in cultured myotubes. These clustered AChRs represent regions of myotube membrane containing high receptor density. We have studied the distribution of the AChR clusters and nuclei to determine whether there is an association in the distribution of nuclei beneath AChR clusters. AChR clusters were visualized with alpha-bungarotoxin conjugated to tetramethylrhodamine (alpha BTX-TMR) and the nuclei were stained with bisbenzimide which binds specifically to DNA. This double label procedure, and the computerized analysis of the data allowed us to determine the distribution of nuclei and AChR clusters in the same myotube. During early stages of myotube development the nuclei formed aggregates which were comprised of 4 to 10 nuclei in close apposition to one another. This association of AChR clusters with nuclear aggregates was greatest at Day 4 after plating. As the number of nuclear aggregates associated with clusters decreased the number of nuclei in the aggregates also decreased and the AChR clusters decreased in size as well as number. At all time points examined, the concentration of myotube nuclei in the cells was 3 to 12 times higher beneath areas of AChR clusters than away from clusters. Our computerized analysis shows that there is an association of the AChR clusters with the nuclear region during myotube development.     

Domain V peptides inhibit beta2-glycoprotein I-mediated mesenteric ischemia/reperfusion-induced tissue damage and inflammation

Reperfusion of ischemic tissue induces significant tissue damage in multiple conditions, including myocardial infarctions, stroke, and transplantation. Although not as common, the mortality rate of mesenteric ischemia/reperfusion (IR) remains >70%. Although complement and naturally occurring Abs are known to mediate significant damage during IR, the target Ags are intracellular molecules. We investigated the role of the serum protein, β2-glycoprotein I as an initiating Ag for Ab recognition and β2-glycoprotein I (β2-GPI) peptides as a therapeutic for mesenteric IR. The time course of β2-GPI binding to the tissue indicated binding and complement activation within 15 min postreperfusion. Treatment of wild-type mice with peptides corresponding to the lipid binding domain V of β2-GPI blocked intestinal injury and inflammation, including cellular influx and cytokine and eicosanoid production. The optimal therapeutic peptide (peptide 296) contained the lysine-rich region of domain V. In addition, damage and most inflammation were also blocked by peptide 305, which overlaps with peptide 296 but does not contain the lysine-rich, phospholipid-binding region. Importantly, peptide 296 retained efficacy after replacement of cysteine residues with serine. In addition, infusion of wild-type serum containing reduced levels of anti-β2-GPI Abs into Rag-1(-/-) mice prevented IR-induced intestinal damage and inflammation. Taken together, these data suggest that the serum protein β2-GPI initiates the IR-induced intestinal damage and inflammatory response and as such is a critical therapeutic target for IR-induced damage and inflammation.     

Dexamethasone alters F-actin architecture and promotes cross-linked actin network formation in human trabecular meshwork tissue

Elevated intraocular pressure is an important risk factor for the development of glaucoma, a leading cause of irreversible blindness. This ocular hypertension is due to increased hydrodynamic resistance to the drainage of aqueous humor through specialized outflow tissues, including the trabecular meshwork (TM) and the endothelial lining of Schlemm's canal. We know that glucocorticoid therapy can cause increased outflow resistance and glaucoma in susceptible individuals, that the cytoskeleton helps regulate aqueous outflow resistance, and that glucocorticoid treatment alters the actin cytoskeleton of cultured TM cells. Our purpose was to characterize the actin cytoskeleton of cells in outflow pathway tissues in situ, to characterize changes in the cytoskeleton due to dexamethasone treatment in situ, and to compare these with changes observed in cell culture. Human ocular anterior segments were perfused with or without 10(-7) M dexamethasone, and F-actin architecture was investigated by confocal laser scanning microscopy. We found that outflow pathway cells contained stress fibers, peripheral actin staining, and occasional actin "tangles." Dexamethasone treatment caused elevated IOP in several eyes and increased overall actin staining, with more actin tangles and the formation of cross-linked actin networks (CLANs). The actin architecture in TM tissues was remarkably similar to that seen in cultured TM cells. Although CLANs have been reported previously in cultured cells, this is the first report of CLANs in tissue. These cytoskeletal changes may be associated with increased aqueous humor outflow resistance after ocular glucocorticoid treatment.