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Basic fibroblast growth factor (bFGF) is an important stimulator of angiogenesis involving in neovascularization progression. The aim of this study is to evaluate whether a liposomal vaccine (MLB) based on xenogeneic human bFGF plus monophosphoryl lipid A (MPLA) could effectively induce cross-reaction immunity in mice and increase antitumor activity. Sera of mice were analyzed and IgG antibody titer in MLB group was obviously higher than other groups including the mice immunized with liposomal bFGF vaccine, bFGF plus Freund's adjuvant, empty liposome and PBS. Furthermore, tumor metastasis was significantly inhibited in MLB group, compared with L and PBS group. The IFN-γ production of cultured splenocytes in vitro was evidently up-regulated meanwhile IL-4 production sustained in a low level, revealing that this vaccine stimulated Th1 immunity response preferentially. Taken together, these findings suggested that this novel bFGF vaccine could effectively induce humoral immunity through cross-reaction, mediate Th1 immune response preferentially and enhance antitumor activity in vivo., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Precursor cell proliferation is present in the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate gyrus of the hippocampus of adult rat and persists during aging although at reduced levels. Previous studies have shown that acute intermittent nicotine treatment significantly increases fibroblast growth factor-2 (FGF-2) expression in several brain regions of aged rats. The aim of the present investigation was to test the hypothesis that nicotine-induced expression of FGF-2 may restore the age-related decline of precursor cell proliferation. It was first demonstrated that nicotine treatment increases both mRNA and protein FGF-2 in the SVZ of aged male rats (18 months old). The effect of nicotine on precursor cell proliferation in the SVZ was studied by i.p. injection of 5-bromo-2'-deoxyuridine (BrdU) 40 mg/kg to label dividing cells. The nicotine treatment was found to significantly enhance precursor cell proliferation in the SVZ. This increase was sufficiently large to restore the age-related decline of proliferating precursor cells observed in aged rats to that found in young adult rats (3 months old). FGF-2 was expressed in GFAP-positive cells and may act via its receptor FGFR1 that was found expressed in nestin-positive cells of the SVZ. The data obtained demonstrated that the age-related decline of precursor cell proliferation may be counteracted by activating a trophic mechanism mediated by FGF-2.

We analyzed in more detail the effect of basic fibroblast growth factor (bFGF) on morphogenesis of rat hippocampal neurons in dissociated cell culture. As a result, we found that bFGF selectively promoted the bifurcation and growth of axonal branches without affecting the elongation rate of primary axons. The dendritic outgrowth was rather inhibited by bFGF. These effects of bFGF resulted in increased complexity of axonal trees. The effect of bFGF was concentration dependent (0.1-10 ng/ml) and was abolished by the presence of anti-bFGF neutralizing antibody. The accelerated axonal branch formation in the presence of bFGF was restored to the basal rate following removal of bFGF, suggesting that the action of bFGF is reversible and that the continuous presence is required for bFGF to accelerate the branch formation. bFGF probably works as a progression signal rather than as a triggering signal. The bFGF-mediated acceleration of axonal branch formation was blocked by treatment with heparitinase and by tyrosine inhibitors, herbimycin A and lavendustin A, indicating the importance of heparan sulfate and tyrosine kinase in bFGF signal transduction. Treatment with a protein kinase C activator phorbol-12-myristate-13-acetate did not significantly affect the neurite branching, and the action of bFGF was not blocked by a protein kinase C inhibitor staurosporine. Protein kinase C is unlikely to play a role in branch formation. The novel action of bFGF as a regulator of axonal branching must be a particularly useful model for the study of neuritogenesis and synaptogenesis of brain neurons.

By means of two monoclonal antibodies specific for heparan sulfate (HS)-related epitopes, one (10E4) against native HS chains and one (3G10) against desaturated uronates, a highly regional and differential distribution of these two epitopes have been observed in the adult rat brain. The 10E4 epitope immunoreactivity (IR) is mainly found in the substantia nigra, the red nucleus and the subgranular zone of the dentate gyrus, while the 3G10 epitope IR is mainly found in the CA2 area of the hippocampal formation and the pyramdial cells in the layer V of the frontoparietal cortex. The codistribution of both types of IRs with basic fibroblast growth factor (bFGF, FGF-2) in neurons and astroglia supports the notion that heparan sulfate proteoglycans (HSPG) in the extracellular matrix may serve as a site for storage of bFGF and assist in the bFGF-induced activation of the high-affinity FGF receptors linked to astroglia and neurons in these discrete areas.

An antiserum against basic fibroblast growth factor (bFGF) was shown to recognize an 18-kDa protein (possibly bFGF) in crude neocortical extracts by immunoblot and used to investigate the changes of bFGF immunoreactivity in neurons and astrocytes of the cerebral cortex of rats 1-21 days after unilateral occlusion of the middle cerebral artery (MCA). The mildly ischemic neocortex exhibited no signs of cell loss or degeneration in Nissl-stained sections 1-14 days after MCA occlusion, but it contained pyramidal cell bodies and processes with more intense bFGF immunoreactivity than did the control neocortex. bFGF immunoreactivity in the ischemic hemisphere gradually declined in intensity and by 21 days after MCA occlusion, it had reached the control level. On the other hand, there were many bFGF immunoreactive astrocytes in the primary olfactory cortex on the side of infarction. These findings suggest that MCA occlusion causes an increase in bFGF content not only in astrocytes but also in neurons, depending on the severity of the ischemic insult in individual cortical regions. The transient augmentation of bFGF expression or accumulation in mildly ischemic pyramidal neurons but not in astrocytes is in line with previous studies suggesting the neurotrophism of exogenously applied bFGF.

A rabbit antiserum (R917) was raised to a purified fraction of bovine brain basic fibroblast growth factor (bFGF). On Western blots of rat midbrain extract, the antiserum did not recognize low molecular weight forms of bFGF. Instead, it recognized a single band of 27-28 kDa. Immunohistochemically, the antiserum preferentially stained a subpopulation of calbindin-negative mesencephalic dopaminergic neurons. The positive somata were mainly packed in a ventral portion of the tegmentum including the A10 region, the ventral tegmental area and the pars compacta of the medial substantia nigra, but were also scattered in both the pars compacta and reticulata portions of the lateral substantia nigra. Processes of dendrites and axons were clearly visible. Terminal fields were located in striosomes, the dorsolateral rim of the neostriatum, the anterodorsal aspect of the nucleus accumbens shell, the infralimbic cortex, and the medial prefrontal cortex. These results suggest that trophic specialization in subpopulations may occur in all three of these dopaminergic projection systems, i.e. the nigrostriatal, mesolimbic and mesocortical pathways.

The immunohistochemical localization of basic fibroblast growth factor (bFGF) was studied in the adult rat brain, using a specific antibody against a synthetic bFGF fragment (the N-terminal 12 residues). Widespread but uneven regional localization of bFGF-like immunoreactive neurons and fibers was observed. Ependymal cells were also stained. The immunoreactive neurons were found in the cerebral cortex, olfactory bulb, septum, basal magnocellular nuclei, thalamus, hypothalamus, globus pallidus, hippocampus, amygdala, red nucleus, central gray of the midbrain, cerebellum, dorsal tegmental area, reticular formation, cranial motor nuclei and spinal cord. Immunoreactive fiber bundles and nerve terminals were also detected. These results indicate that bFGF is produced by or present in a specific neuronal cell population of the central nervous system.

The distribution of bFGF in mouse and rat retinas was established using an antibody generated against a rat specific bFGF sequence. The patterns established with this antibody were distinctly different in comparison to the immunostaining patterns using antibodies prepared against similar regions of bovine and human bFGF sequences. Using the anti-rat antibody, at postpartum day 5, both species demonstrate bFGF-like labeling in the outer retina, primarily associated with developing photoreceptors. The anti-rat bFGF antibody in the adult mouse produces intense labeling at the level of the photoreceptor outer segment, whereas in the adult rat, Müller's cells and RPE were intensely labeled but the photoreceptors appeared to be unlabeled. In contrast, the bovine and human antibodies consistently label ganglion cells in both mouse and rat retinas, cells in the inner aspect of the inner nuclear layer and developing horizontal cells, whereas no photoreceptor labeling was observed. These results suggest that minor species differences in a short segment of the bFGF molecule used to generate these antibodies may result in major differences in apparent bFGF-like immunolocalization.

When dissociated subcortical cells were cultured in the presence of conditioned medium of relatively differentiated astrocytes (ACM), a marked increase was observed in the expression of choline acetyltransferase (ChAT), an enzyme required for the synthesis of the neurotransmitter acetylcholine. Astrocytes from the target regions of subcortical neurons, the hippocampus and the cerebral cortex, produced neurotrophic factor consistently more than those derived from the nontarget region, the cerebellum. The production of cholinergic trophic activity was increased with the maturation of astrocytes. Even though, nerve growth factor (NGF) and ciliary neurotrophic factor (CNTF) are known cholinergic trophic compounds produced by astrocytes in vitro, a large part of the neurotrophic activity in our ACM was not related to either of these 2 factors. This is because (i) ACM and NGF produced an additive effect on ChAT activity, (ii) only a small proportion of the cholinergic trophic activity in ACM was abolished by anti-NGF antibody, and (iii) treatment with CNTF had no effect on ChAT activity of basal forebrain cholinergic neurons. On the other hand, when cholinergic neurons are cultured on a preformed layer of astrocytes, addition of basal fibroblast growth factor (bFGF) failed to increase further the ChAT activity. Similarly the effects of ACM and bFGF were not additive. A large proportion of the cholinergic trophic activity in ACM was neutralized by anti-bFGF antibody. These findings would suggest that the trophic activity on septal cholinergic neurons in our ACM was due to bFGF or a bFGF-like compound.

When neurons die, the filaments of neurofibrillary tangles (NFT) undergo structural and antigenic modifications. The exact mechanism of this modification is unknown, but glial cells could play an important role. Previous studies have shown that astroglial processes infiltrate extracellular NFT. In this study we use double immunolabelling to show that microglia also infiltrate extracellular NFT. Therefore, along with the previously identified astroglia, the microglia could be responsible for the modification of extracellular NFT.