In vivo inhibition of lens regrowth by fibroblast growth factor 2-saporin.

PURPOSE: To investigate the ability of fibroblast growth factor (FGF) 2-saporin to prevent lens regrowth in the rabbit. METHODS: Chemically conjugated and genetically fused FGF2-saporin (made in Escherichia coli) were used. Extracapsular extraction of the lens was performed on the rabbit, and the cytotoxin either was injected directly into the capsule bag or was administered by FGF2-saporin-coated, heparin surface-modified (HSM) polymethylmethacrylate intraocular lenses. The potential of the conjugate was checked by slit lamp evaluation of capsular opacification and by measuring crystallin synthesis. Toxin diffusion and sites of toxin binding were assessed by immunohistochemistry. Possible toxicity was determined by histologic analysis of ocular tissues. RESULTS: FGF2-saporin effectively inhibited lens regrowth when it was injected directly into the capsular bag. However, high concentration of the toxin induced transient corneal edema and loss of pigment in the iris. Intraocular lenses coated with FGF2-saporin reduced lens regrowth and crystallin synthesis without any detectable clinical side effect. After implantation, FGF2-saporin was shown to have bound to the capsules and, to a lesser extent, to the iris; no histologic damage was found on ocular tissues as a result of implantation of drug-loaded HSM intraocular lenses. CONCLUSIONS: Chemically conjugated (FGF2-SAP) and genetically fused FGF2-saporin (rFGF2-SAP) bound to HSM intraocular lenses can prevent lens regrowth in the rabbit.

Peroxynitrite cytotoxicity on bovine retinal pigmented epithelial cells in culture.

Peroxynitrite induced in vitro a dose dependent toxicity on retinal pigmented epithelial (RPE) cells. Cell death was partially mediated by apoptosis as demonstrated by nuclear fragmentation and TdT-mediated dUTP nick-end labeling assay. Peroxynitrite-induced tyrosine nitration was revealed by immunocytochemistry, both in the cytoplasm and in the nucleus of the cells. Nitration was not observed in RPE cells, producing nitric oxide (NO) after stimulation by lipopolysacharide and interferon-g (IFN-gamma), suggesting that peroxynitrite was not formed in vitro in such conditions. Peroxynitrite could be responsible for the retinal damages observed in pathological conditions in which NO has been demonstrated to be involved. In this context, EGb761, identified as a free radical scavenger, was showed herein to protect RPE cells against peroxynitrite injury.

Microglial responsiveness as a sensitive marker for trimethyltin (TMT) neurotoxicity.

Activation of microglia is a well-documented phenomenon associated with diverse pathological conditions of the central nervous system. In order to investigate the involvement of microglial cells in the neurotoxic action of the heavy metal compound trimethyltin, three-dimensional brain cell cultures were treated during an early developmental period, using concentrations at or below the limit of cytotoxicity. Microglial cells were studied by cytochemical staining, using horseradish peroxidase-conjugated B4 isolectin (GSI-B4). In parallel, neurotoxic effects were assessed by determining the content of synaptophysin and synapsin I, both in the total homogenates and in the synaptosomal fraction of the cultures. Changes in the content of the specific growth cone protein, GAP-43, were also analyzed. It was found that low, non-cytotoxic concentrations of TMT (10(-9) to 10(-8) M) caused a significant increase in the number and/or the clustering of microglial cells. A decrease in the synaptic protein (synapsin I, synaptophysin) content was detected at 10(-8) M of TMT in synaptosomal fractions, whereas in the total homogenates, changes in synaptic proteins and GAP-43 were observed only at the cytotoxic TMT concentration (10(-6) M). Although it remains to be shown whether the microglial response is caused by direct or indirect action of TMT, the present findings show that microglial responsiveness can be detected prior to any sign of neuronal degeneration, and may serve as a sensitive indicator for heavy metal neurotoxicity in the brain.

Fatty acids and retinoids control lipid metabolism through activation of peroxisome proliferator-activated receptor-retinoid X receptor heterodimers.

The nuclear hormone receptors called PPARs (peroxisome proliferator-activated receptors alpha, beta, and gamma) regulate the peroxisomal beta-oxidation of fatty acids by induction of the acyl-CoA oxidase gene that encodes the rate-limiting enzyme of the pathway. Gel retardation and cotransfection assays revealed that PPAR alpha heterodimerizes with retinoid X receptor beta (RXR beta; RXR is the receptor for 9-cis-retinoic acid) and that the two receptors cooperate for the activation of the acyl-CoA oxidase gene promoter. The strongest stimulation of this promoter was obtained when both receptors were exposed simultaneously to their cognate activators. Furthermore, we show that natural fatty acids, and especially polyunsaturated fatty acids, activate PPARs as potently as does the hypolipidemic drug Wy 14,643, the most effective activator known so far. Moreover, we discovered that the synthetic arachidonic acid analogue 5,8,11,14-eicosatetraynoic acid is 100 times more effective than Wy 14,643 in the activation of PPAR alpha. In conclusion, our data demonstrate a convergence of the PPAR and RXR signaling pathways in the regulation of the peroxisomal beta-oxidation of fatty acids by fatty acids and retinoids.

The role of PKCzeta in amikacin-induced apoptosis in the cochlea: prevention by aspirin.

Aminoglycoside antibiotics are ototoxic, inducing irreversible sensorineural hearing loss mediated by oxidative and excitotoxic stresses. The NF-kappaB pathway is involved in the response to aminoglycoside damage in the cochlea. However, the molecular mechanisms of this ototoxicity remain unclear. We investigated the expression of PKCzeta, a key regulator of NF-kappaB activation, in response to aminoglycoside treatment. Amikacin induced PKCzeta cleavage and nuclear translocation. These events were concomitant with chromatin condensation and paralleled the decrease in NF-kappaB (p65) levels in the nucleus. Amikacin also induced the nuclear translocation of apoptotic inducing factor (AIF). Prior treatment with aspirin prevented PKCzeta cleavage and nuclear translocation. Thus, aspirin counteracts the early effects of amikacin, thereby protecting hair cells and spiral ganglion neurons. These results demonstrate that PKCzeta acts as sentinel connecting specific survival pathways to mediate cellular responses to amikacin ototoxicity.

ER stress activates the NLRP3 inflammasome via an UPR-independent pathway.

Uncontrolled endoplasmic reticulum (ER) stress responses are proposed to contribute to the pathology of chronic inflammatory diseases such as type 2 diabetes or atherosclerosis. However, the connection between ER stress and inflammation remains largely unexplored. Here, we show that ER stress causes activation of the NLRP3 inflammasome, with subsequent release of the pro-inflammatory cytokine interleukin-1β. This ER-triggered proinflammatory signal shares the same requirement for reactive oxygen species production and potassium efflux compared with other known NLRP3 inflammasome activators, but is independent of the classical unfolded protein response (UPR). We thus propose that the NLRP3 inflammasome senses and responds to ER stress downstream of a previously uncharacterized ER stress response signaling pathway distinct from the UPR, thus providing mechanistic insight to the link between ER stress and chronic inflammatory diseases.

Expression of O⁶-methylguanine-DNA methyltransferase in childhood medulloblastoma.

Medulloblastomas (MB) are the most common malignant brain tumors in childhood. Alkylator-based drugs are effective agents in the treatment of patients with MB. In several tumors, including malignant glioma, elevated O(6)-methylguanine-DNA methyltransferase (MGMT) expression levels or lack of MGMT promoter methylation have been found to be associated with resistance to alkylating chemotherapeutic agents such as temozolomide (TMZ). In this study, we examined the MGMT status of MB and central nervous system primitive neuroectodermal tumor (PNET) cells and two large sets of primary MB. In seven MB/PNET cell lines investigated, MGMT promoter methylation was detected only in D425 human MB cells as assayed by the qualitative methylation-specific PCR and the more quantitative pyrosequencing assay. In D425 human MB cells, MGMT mRNA and protein expression was clearly lower when compared with the MGMT expression in the other MB/PNET cell lines. In MB/PNET cells, sensitivity towards TMZ and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) correlated with MGMT methylation and MGMT mRNA expression. Pyrosequencing in 67 primary MB samples revealed a mean percentage of MGMT methylation of 3.7-92% (mean: 13.25%, median: 10.67%). Percentage of MGMT methylation and MGMT mRNA expression as determined by quantitative RT-PCR correlated inversely (n = 46; Pearson correlation r (2) = 0.14, P = 0.01). We then analyzed MGMT mRNA expression in a second set of 47 formalin-fixed paraffin-embedded primary MB samples from clinically well-documented patients treated within the prospective randomized multicenter trial HIT'91. No association was found between MGMT mRNA expression and progression-free or overall survival. Therefore, it is not currently recommended to use MGMT mRNA expression analysis to determine who should receive alkylating agents and who should not.

Immunization of BALB/c mice with Helicobacter urease B induces a T helper 2 response absent in Helicobacter infection.

BACKGROUND & AIMS: Infection with Helicobacter induces a T helper type 1 response in mice and humans. Mice can be cured or protected from infection with Helicobacter by mucosal immunization with recombinant H. pylori urease B subunit (rUreB). This study characterizes the immune response of infected mice immunized with rUreB. METHODS: BALB/c mice were infected with H. felis. Two weeks later, they were orally immunized four times with rUreB and cholera toxin (CT) at weekly intervals. Controls were only infected or sham-immunized with CT. Animals were killed at various times after immunization. Splenic CD4(+) cells were obtained and cultured in vitro with rUreB to evaluate antigen-specific proliferation and induction of interferon gamma and interleukin 4 secretion. RESULTS: All rUreB-immunized mice (n = 8) were cured from infection 3 weeks after the fourth immunization. Immunization induced a proliferative response of splenic CD4(+) cells, a progressive decrease in interferon gamma secretion, and a concomitant increase in interleukin 4 secretion after each immunization. A simultaneous increase in rUreB specific serum immunoglobulin G1 levels was observed in infected/immunized mice. CONCLUSIONS: In BALB/c mice, therapeutic mucosal immunization with rUreB induces progressively a Th2 CD4(+) T cell response resulting in the elimination of the pathogen.

Induction of apoptosis by mercury compounds depends on maturation and is not associated with microglial activation

The earliest sign of neurotoxicity observed after exposure of three-dimensional brain cell cultures to low concentrations of mercury compounds is a microglial reaction. We hypothesized that an induction of apoptosis by mercury compounds could be an activating signal of the microglial reaction. Aggregating brain cell cultures of fetal rat telencephalon were treated for 10 days with either mercury chloride or monomethylmercury chloride at noncytotoxic concentrations during two developmental periods: from day 5 to 15, corresponding to an immature stage, and from day 25 to 35 corresponding to a mature stage. Apoptosis was evaluated by the TUNEL technique. It was found that both mercury compounds caused a significant increase in the number of apoptotic cells, but exclusively in immature cultures exhibiting also spontaneous apoptosis. Double staining by the TUNEL technique combined with either neuronal or astroglial markers revealed that the proportion of cells undergoing apoptosis was highest for astrocytes. Furthermore neither an association nor a colocalization was found between apoptotic cells and microglial cells. In conclusion, it appears that the induction of apoptosis by mercury compounds in immature cells is only an acceleration of a spontaneously occurring process, and that it is not a directly related to the early microglial reaction.

In vitro characterization of immune-related properties of human fetal bone cells for potential tissue engineering applications.

We describe herein some immunological properties of human fetal bone cells recently tested for bone tissue-engineering applications. Adult mesenchymal stem cells (MSCs) and osteoblasts were included in the study for comparison. Surface markers involved in bone metabolism and immune recognition were analyzed using flow cytometry before and after differentiation or treatment with cytokines. Immunomodulatory properties were studied on activated peripheral blood mononuclear cells (PBMCs). The immuno-profile of fetal bone cells was further investigated at the gene expression level. Fetal bone cells and adult MSCs were positive for Stro-1, alkaline phosphatase, CD10, CD44, CD54, and beta2-microglobulin, but human leukocyte antigen (HLA)-I and CD80 were less present than on adult osteoblasts. All cells were negative for HLA-II. Treatment with recombinant human interferon gamma increased the presence of HLA-I in adult cells much more than in fetal cells. In the presence of activated PBMCs, fetal cells had antiproliferative effects, although with patterns not always comparable with those of adult MSCs and osteoblasts. Because of the immunological profile, and with their more-differentiated phenotype than of stem cells, fetal bone cells present an interesting potential for allogeneic cell source in tissue-engineering applications.

Antibacterial burst-release from minimal Ag-containing plasma polymer coatings.

Biomaterials releasing silver (Ag) are of interest because of their ability to inhibit pathogenic bacteria including antibiotic-resistant strains. In order to investigate the potential of nanometre-thick Ag polymer (Ag/amino-hydrocarbon) nanocomposite plasma coatings, we studied a comprehensive range of factors such as the plasma deposition process and Ag cation release as well as the antibacterial and cytocompatible properties. The nanocomposite coatings released most bound Ag within the first day of immersion in water yielding an antibacterial burst. The release kinetics correlated with the inhibitory effects on the pathogens Pseudomonas aeruginosa or Staphylococcus aureus and on animal cells that were in contact with these coatings. We identified a unique range of Ag content that provided an effective antibacterial peak release, followed by cytocompatible conditions soon thereafter. The control of the in situ growth conditions for Ag nanoparticles in the polymer matrix offers the possibility to produce customized coatings that initially release sufficient quantities of Ag ions to produce a strong adjacent antibacterial effect, and at the same time exhibit a rapidly decaying Ag content to provide surface cytocompatibility within hours/days. This approach seems to be favourable with respect to implant surfaces and possible Ag-resistance/tolerance built-up.

IL-4 instructs TH1 responses and resistance to Leishmania major in susceptible BALB/c mice.

Immunity to infection with intracellular pathogens is regulated by interleukin 12 (IL-12), which mediates protective T helper type 1 (TH1) responses, or IL-4, which induces TH2 cells and susceptibility. Paradoxically, we show here that when present during the initial activation of dendritic cells (DCs) by infectious agents, IL-4 instructed DCs to produce IL-12 and promote TH1 development. This TH1 response established resistance to Leishmania major in susceptible BALB/c mice. When present later, during the period of T cell priming, IL-4 induced TH2 differentiation and progressive leishmaniasis in resistant mice. Because immune responses developed via the consecutive activation of DCs and then T cells, the contrasting effects of IL-4 on DC development and T cell differentiation led to immune responses that had opposing functional phenotypes.

Controlled delivery of 5-chlorouracil using poly(ortho esters) in filtering surgery for glaucoma.

PURPOSE: To evaluate the antimitotic and toxic effects of 5-chlorouracil (5-CU) and 5-fluorouracil (5-FU) and study their potential to delay filtering bleb closure in the rabbit eye when released by poly(ortho esters) (POE). METHODS: Rabbit Tenon fibroblasts and human conjunctival cells were incubated with various 5-CU and 5-FU concentrations. Antiproliferative effects and toxicity were evaluated at 24 and 72 hours by monotetrazolium, neutral red, and Hoechst tests and cell counting. Mechanisms of cell death were evaluated using TUNEL assay, annexin V binding, immunohistochemistry for anti-apoptosis-inducing factor (AIF) and LEI/L-DNase II. Trabeculectomy was performed in pigmented rabbits. Two hundred microliters of POE loaded with 1% wt/wt 5-FU or 5-CU was injected into the subconjunctival space after surgery. Intraocular pressure (IOP) and bleb persistence were monitored for 150 days. RESULTS: In vitro, 5-FU showed a higher antiproliferative effect and a more toxic effect than 5-CU. 5-FU induced cell necrosis, whereas 5-CU induced mostly apoptosis. The apoptosis induced by 5-CU was driven through a non-caspase-dependent pathway involving AIF and LEI/L-DNase II. In vivo, at 34 days after surgery, the mean IOP in the POE/5-CU-treated group was 83% of the baseline level and only 40% in the POE/5-FU-treated group. At 100 days after surgery, IOP was still decreased in the POE/5-CU group when compared with the controls and still inferior to the preoperative value. The mean long-term IOP, with all time points considered, was significantly (P < 0.0001) decreased in the POE/5-CU-treated group (6.0 +/- 2.4 mm Hg) when compared with both control groups, the trabeculectomy alone group (7.6 +/- 2.9 mm Hg), and the POE alone group (7.5 +/- 2.6 mm Hg). Histologic analysis showed evidence of functioning blebs in the POE-5-CU-treated eyes along with a preserved structure of the conjunctiva epithelium. CONCLUSIONS: The slow release of 5-CU from POE has a longstanding effect on the decrease of IOP after glaucoma-filtering surgery in the rabbit eye. Thus, the slow release of POE/5-CU may be beneficial for the prevention of bleb closure in patients who undergo complicated trabeculectomy.

Fluorodeoxyuridine improves imaging of human glioblastoma xenografts with radiolabeled iododeoxyuridine.

Use of radiolabeled nucleotides for tumor imaging is hampered by rapid in vivo degradation and low DNA-incorporation rates. We evaluated whether blocking of thymidine (dThd) synthesis by 5-fluoro-2'-deoxyuridine (FdUrd) could improve scintigraphy with radio-dThd analogues, such as 5-iodo-2'-deoxyuridine (IdUrd). We first show in vitro that coincubation with FdUrd substantially increased incorporation of [125I]IdUrd and [3H]dThd in the three tested human glioblastoma lines. Flow cytometry analysis showed that a short coincubation with FdUrd (1 h) produces a signal increase per labeled cell. We then measured biodistribution 24 h after i.v. injection of [125I]IdUrd in nude mice s.c. xenografted with the three glioblastoma lines. Compared with animals given [125I]IdUrd alone, i.v. preadministration for 1 h of 10 mg/kg FdUrd increased the uptake of [125I]IdUrd in the three tumors 4.8-6.8-fold. Compatible with previous reports, there were no side effects in mice observed for 2 months after receiving such a treatment. The tumor uptake of [125I]IdUrd was increased < or =13.6-fold when FdUrd preadministration was stepwise reduced to 1.1 mg/kg. Uptake increases remained lower (between 1.7- and 5.8-fold) in normal proliferating tissues (i.e., bone marrow, spleen, and intestine) and negligible in quiescent tissues. DNA extraction showed that 72-80% of radioactivity in tumor and intestine was bound to DNA. Scintigraphy of xenografted mice was performed at different times after i.v. injection of 3.7 MBq [125I]IdUrd. Tumor detection was significantly improved after FdUrd preadministration while still equivocal after 24 h in mice given [125I]IdUrd alone. Furthermore, background activity could be greatly reduced by p.o. administration of KClO4 in addition to potassium iodide. We conclude that FdUrd preadministration may improve positron or single photon emission tomography with cell division tracers, such as radio-IdUrd and possibly other dThd analogues.