Abstract. Immunohistochemical methods were used to study the distribution of basic FGF in the 18-d rat fetus. The results reveal a pattern of widespread yet specific staining that is consistent with the wide distribution of basic FGE Immunoreactive basic FGF is associated with mesenchymal structures, mesodermand neuroectoderm-derived cells, and their extracellular matrices. As an example, skeletal and smooth muscle cells are strongly positive. The basement membrane underlying the epithelia always contain basic FGE In some tissues (i.e., cartilage and bone) the intensity of immunostaining is dependent on the stage of cell differentiation.Although the staining of tissues is primarily associated with the extracellular matrix, there is significant intracellular staining in various cell types. This is particularly evident in the endocrine cells of the adrenal cortex, testis, and ovary. The histochemical findings reported here support the notion that basic FGF has the characteristics required to mediate many of the effects of the mesenchyme on cell growth and differentiation. The significance of these findings in understanding the role of basic FGF in regulating cell proliferation and differentiation is discussed.

Traumatic injury to the CNS initiates transient and unsuccessful regeneration of damaged neural pathways, accompanied by reactive gliosis, angiogenesis, and deposition of a dense fibrous glial/meningeal scar at the wound site. Basic fibroblast growth factor (basic FGF) is a CNS protein with potent effects on neurons, glia, fibroblasts, and vascular endothelial cells. Hybridization and immunocytochemical methods were used to examine temporal and spatial changes in distribution and levels of basic FGF protein and mRNA and also of its receptor mRNA (flg), following a defined wound to the cerebral cortex of adult rat brains. In the injured brain, a rapid, transient increase in basic FGF mRNA and protein is readily detectable within 7 d of surgery and thereafter declines in the tissues bordering the lesion. The increased expression is localized to multiple cell types including macrophages, neurons, astrocytes, and vascular endothelial cells. The changes in immunoreactive basic FGF parallel changes in the bioactivity of extracted heparin-binding proteins, which include basic FGF. Focal increases in flg mRNA appear 7 d after injury and subside by 14 d. The changes in local basic FGF synthesis, concentration, localization, and bioactivity suggest that this growth factor may contribute to the cascade of cellular events that occur in CNS wound repair.

The single copy gene for human basic fibroblast growth factor (bFGF) has been shown to encode not one but multiple proteins of 24, 23, 22 and 18 kD. Although bioactivities of the 18 kD protein are currently used to define bFGF gene function, it is not yet known if the three larger proteins have these same bioactivities or whether they will serve to define new bFGF gene functions. In this report we present a comparative study describing the de novo synthesis, transport, processing and intracellular location of individual bFGF isoforms. Data from cDNA mutagenesis and COS cell expression experiments show that individual isoforms are differentially localized to either the cell surface or to the nucleus. The 24, 23 and 22 kD proteins (CUG-mediated initiation) exclusively localize in the nucleus while the 18 kD protein (AUG-mediated initiation) is preferentially exported onto the cell surface, but is not released into the surrounding culture medium. Specific CUG or AUG translation initiation codons are necessary and sufficient for the synthesis of each isoform examined and thereby, indirectly, mediate differential localization. Since bFGF does not contain the characteristic signals predicted for cell surface or nuclear targeting, our continuing studies will either unmask its functionally equivalent domain(s) or will identify the requisite participation of yet unknown cellular components.

Maternal cytokines may play instructive roles in development of the neonatal immune system. However, cytokines in colostrum and milk and their transfer from mothers to neonates have not been well documented, except for TGF-beta. Swine provide a unique model to study lactogenic cytokines because the sow's impermeable placenta prohibits transplacental passage. We investigated IL-6 and TNF-alpha (pro-inflammatory), IFN-gamma and IL-12 (Th1), IL-10 and IL-4 (Th2) and TGF-beta1 (Th3) concentrations in sow serum and colostrum/milk and serum of their suckling and weaned piglets and in age-matched colostrum-deprived gnotobiotic piglets. All cytokines were detected in colostrum/milk and correlated with concentrations in sow serum except for mammary-derived TNF-alpha and TGF-beta1. Detection of IL-12 and TGF-beta1 in pre-suckling and colostrum-deprived gnotobiotic piglet serum suggests constitutive production: other cytokines were undetectable confirming absence of transplacental transfer. Peak median cytokine concentrations in suckling piglet serum occurred at post-partum days 1-2 (IL-4>IL-6>IFN-gamma>IL-10). The effects in vitro of physiologically relevant concentrations of the two predominant lactogenic cytokines (TGF-beta1 and IL-4) on porcine naive B cell responses to lipopolysaccharide (LPS) and rotavirus (RV) were investigated. High (10 ng/ml) TGF-beta1 suppressed immunoglobulin secreting cell responses to LPS and rotavirus; low concentrations (0.1 ng/ml) promoted isotype switching to IgA antibody. Interleukin-4 induced inverse dose-dependent (0.1 ng>10 ng/ml) isotype switching to IgA and enhanced IgM secreting cell responses to LPS and rotavirus. In summary, we documented the transfer and persistence of maternal cytokines from colostrum/milk to neonates and their potential role in Th-2 biased IgA responses and reduced immunologic responsiveness of neonates.

It is becoming clear that transforming growth factor beta (TGF beta) may be a key factor regulating inflammatory and tissue specific wound responses. Because the formation of a glial-collagen scar at CNS lesion sites is thought to contribute to the pathology associated with penetrating CNS injuries, and because in the periphery TGF beta 1 stimulates fibroblast deposition of scar tissue, we used in situ hybridization and immunohistochemistry to investigate the effect of a defined cerebral lesion on the local expression of TGF beta 1. Induction of TGF beta 1 mRNA and protein is relatively diffuse in the neuropile around the margins of the lesion at 1, 2 and 3 days, but becomes localized to the region of the glial scar at 7 and 14 days. The signal intensity for TGF beta 1 mRNA and protein is maximal between 2 and 3 days and decreases between 7 and 14 days after lesion. The predominant cell types in the neuropile localizing TGF beta 1 mRNA and protein have the morphological characteristics of astrocytes, although macrophages are also detected. An induction of TGF beta 1 mRNA was also observed in endothelial cells of the meninges, hippocampal fissure and choroid plexus, at 2 and 3 days. However, this is dramatically reduced by 7 days and has disappeared by 14 days. These results suggest a role for TGF beta 1, not only in inflammation, but also in the tissue-specific glial scar formation that occurs in the CNS. Furthermore, they suggest a potential therapeutic use of TGF beta 1 antagonists in the CNS to help limit the pathogenesis associated with matrix deposition in the wound.