Reduction in basic fibroblast growth factor mediated angiogenesis in vivo by linomide.

Linomide (N-phenylmethyl-1,2-dihydro-4-hydroxyl-1-methyl-2-oxoquinoline-3-carboxa mide) is a novel immunomodulator with a potent anti-tumoral activity. This study was undertaken to test the effect of Linomide on basic fibroblast growth factor (bFGF) induced angiogenesis in vivo, which manifests itself in an increased number of blood vessels per unit of cell infiltrated area. Subcutaneously implanted polyvinyl alcohol sponges (PVS) in guinea pigs were used as a model system to quantitate angiogenesis in vivo. Oral treatment with Linomide was able to reduce significantly the bFGF induced blood vessel growth and proliferation within the implanted PVS, relative to untreated controls. In addition, Linomide significantly reduced the bFGF mediated augmentation of protein and collagen content in the implanted PVS, indicating an inhibition in the deposition of extracellular matrix (ECM). We conclude that the potent inhibition of bFGF induced angiogenesis by Linomide in vivo in addition to immunomodulatory effects may have potentially important clinical applications.

Thalidomide reduces vascular density in granulation tissue of subcutaneously implanted polyvinyl alcohol sponges in guinea pigs.

The efficacy of thalidomide in the treatment of erythema nodosum leprosum is a well established fact; there is also accumulating evidence of its therapeutic value in a number of other inflammatory and immune-mediated conditions. In addition, thalidomide has been shown to be an inhibitor of angiogenesis induced by basic fibroblast growth factor (bFGF). Nevertheless, its mechanism of action remains speculative. Using guinea pigs, orally administered thalidomide significantly enhanced the response of multinucleated foreign body giant cells (p<0.05) in subcutaneously implanted polyvinyl alcohol sponges. Furthermore, the drug exerted a dual effect in that it reduced vascular density (p<0.05), which was not abolished by recombinant human bFGF, and at the same time amplified the granulomatous response with and without bFGF (p<0.05 and p<0.01, respectively). The results of our experiments represent a further step toward understanding the mechanism of action of thalidomide, with implications for its potential use in wound healing and scar formation as well as in the control of tumorigenesis.

Exogenous non-crosslinked collagen enhances granulation tissue formation in dermal excision wounds in guinea pigs.

Based on previous observations indicating a role for collagen peptides in eliciting a positive feedback for collagen biosynthesis, this study was initiated to elucidate the effect of non-crosslinked collagen on granulation tissue formation in dermal excision wounds. The wounds were treated with either non-crosslinked or crosslinked native collagen, or left untreated as controls. Granulation tissue was analyzed for collagen type I mRNA, for levels of interstitial collagen and for the number of blood vessels. The results indicated significant increases in procollagen type I mRNA, in interstitial collagen, in the number of blood vessels and in epithelial advance in the non-crosslinked collagen-treated wounds relative to the untreated controls. It is assumed that the presence of non-crosslinked collagen in a healing wound enhances both procollagen type I biosynthesis and the repair process of dermal wounds, due to the more readily released collagen peptides derived from this exogenous collagen dressing.

Reduction in pulmonary fibrosis in vivo by halofuginone.

Pulmonary fibrosis is a disorder causing a high mortality rate for which therapeutic options are limited. Therefore, the effect of halofuginone, a novel inhibitor of collagen type I synthesis, on bleomycin-induced pulmonary fibrosis was studied in rats. Pulmonary fibrosis was induced by intraperitoneal injections of bleomycin for seven consecutive days, and halofuginone was administered intraperitoneally every second day during the entire experimental period of 42 d. Collagen determination in the lungs and the examination of histologic sections showed that halofuginone significantly reduced fibrosis relative to the untreated control rats. We conclude that halofuginone is a potent in vivo inhibitor of bleomycin-induced pulmonary fibrosis, and that it may potentially be used as a novel therapeutic agent for the treatment of this dysfunction.

Control of p62 binding to TGN38/41 by phosphorylation.

TGN38/41 cycles between the trans-Golgi network (TGN) and plasma membrane, traversing three sorting compartments: the TGN, plasma membrane and early endosome. The targeting signals responsible for this complex itinerary reside in a short cytoplasmic domain of 33 amino acid residues. We show that phosphorylation of the cytoplasmic domain of TGN38 prevents binding of p62--a cytoplasmic protein essential for exocytic vesicle formation. Thus the cycle of TGN38/41 traffic, and by implication the pathway of exocytosis, could be controlled by phosphorylation of the TGN38 cytoplasmic domain.

Phosphoglycerate kinase pseudogenes in the tammar wallaby and other macropodid marsupials.

Phosphoglycerate kinase (EC 2.7.2.3; PGK) exists in two forms in marsupials. PGK1 is an X-linked house-keeping enzyme, and PGK2 is a mainly testis-specific enzyme under autosomal control. We have used PGK1 probes derived from two closely related species of macropodid marsupials (kangaroos and wallabies) to demonstrate the existence of a large family of pseudogenes in the tammar wallaby (Macropus eugenii). Over 30 fragments are detectable after Taq digestion. We estimate that there are 25-30 copies per genome. Most are autosomally inherited and are apparently not closely linked. Only two restriction fragments that appeared to be sex linked could be detected. Varying degrees of hybridization of fragments to the probes suggest different levels of homology, and hence different ages of origin. The existence of two PGK1 homologous restriction fragments from the X and a large number from the autosomes was also demonstrated by somatic cell hybridization for two other macropodid species, the wallaroo (M. robustus) and the red kangaroo (M. rufus). These results are compared with those from human and mouse, and it is suggested that the propensity of PGK1 to form pseudogenes is an ancient (approximately 130 MYR BP) characteristic of mammals. The high level of polymorphism detected in the tammar makes these PGK1 probes potentially useful for measuring genetic variability in this species and other macropodids.

PCR derived cDNA clones for X-linked phosphoglycerate kinase-1 in a marsupial, the tammar wallaby (Macropus eugenii).

cDNA clones for the X-linked PGK-1 were obtained from a tammar wallaby liver by PCR and sequenced. The PGK-1 gene published here is the consensus sequence of those clones. The sequence represents an open reading frame of 1251 bp. Sequence comparisons to X-linked and autosomal sequences showed the greatest homology with the X-linked PGK-1 genes in eutherian species. This sequence opens the way for studying the paternal X inactivation phenomenon in marsupials and will assist in defining the time course of mammalian evolution.

Control of primary and secondary antibody responses by cytotoxic T lymphocytes specific for a soluble antigen.

BALB/c mice immunized with keyhole limpet hemocyanin (KLH) develop Thy-1+ and CD8+, KLH-specific cytotoxic T cells (Tc). Such Tc cells can lyse TNP-specific B cells activated with TNP-KLH, but not with TNP-ovalbumin. Cytotoxicity was inhibited by anti-H-2K/D antibodies, but not by anti-Ia antibodies, suggesting a major histocompatibility complex class I-restricted killing. Selective enrichment for virgin and memory TNP-specific B cells revealed that the latter cells were relatively resistant to lysis by KLH-specific Tc cells. Depletion of CD8+ T cells from cultures of TNP-specific virgin B cells activated with TNP-KLH and KLH-primed T cells, increased the titer of anti-TNP antibodies in the culture supernatants. This increase was reduced if KLH-primed CD8+ T cells were added to the culture 1 day before its termination. Anti-TNP antibody secretion by memory B cells activated in the same manner was not affected by depletion or addition of CD8+ cells. These results suggest that Tc cells are generated following immunization with a soluble antigen which may participate in the down-regulation of primary B cell responses.