Identification of Mechanism and Pathway of the Interaction between the African Traditional Medicine, Sutherlandia Frutescens, and the Antiretroviral Protease Inhibitor, Atazanavir, in Human Subjects Using Population Pharmacokinetic (PK) Analysis.

Although the use of the indigenous Southern African plant, Sutherlandia frutescens (SF) for the treatment of HIV/AIDS has previously been described, the risk which it may pose to the safety and efficacy of ARVs and the potential mechanisms which underlie such effects may have clinical significance and relevance. The protease inhibitor (PI), atazanavir (ATV) is a substrate of the efflux transporter, P-gp which modulates absorption in the small intestine, as well as CYP3A4 and CYP3A5enzymes which facilitate metabolism in the small intestine and liver. The objective of this study was to investigate the effect of SF on the pharmacokinetics (PK) of atazanavir (ATV) and to use a population PK analysis to fit and explain plasma concentration vs. time profiles of ATV generated in a previously conducted study in healthy male subjects in order to understand and postulate on the potential mechanism(s) of the drug-drug interaction. The population PK Compartmental Analysis of ATV before and after a two-week regimen of Phyto Nova Sutherlandia SU1 tablets which contain SF plant material indicated that a two compartment model with a dual absorption mechanism best explained the data. The dual absorption mechanism is hypothesized to reflect "passive" (first-order, Ka parameter) and "active" (zero-order, K0 parameter) absorption processes. The model suggested that the mechanism by which SF reduced the overall bioavailability of ATV may be modulated via the inhibition of the "active" absorption process. This study has highlighted the utility of population PK analyses in postulating probable mechanism(s) whereby an ATM or a herbal medicine interacts with an allopathic drug.

Imaging atherosclerotic plaque inflammation via folate receptor targeting using a novel 18F-folate radiotracer.

Folate receptor ß (FR-ß) is overexpressed on activated, but not resting, macrophages involved in a variety of inflammatory and autoimmune diseases. A pivotal step in atherogenesis is the subendothelial accumulation of macrophages. In nascent lesions, they coordinate the scavenging of lipids and cellular debris to define the likelihood of plaque inflammation and eventually rupture. In this study, we determined the presence of FR-ß-expressing macrophages in atherosclerotic lesions by the use of a fluorine-18-labeled folate-based radiotracer. Human endarterectomized specimens were used to measure gene expression levels of FR-ß and CD68. Increased FR-ß and CD68 levels were found in atherosclerotic plaques compared to normal artery walls by quantitative real-time polymerase chain reaction. Western blotting and immunohistochemistry demonstrated prominent FR-ß protein levels in plaques. FR-ß-positive cells colocalized with activated macrophages (CD68) in plaque tissue. Carotid sections incubated with 3'-aza-2'-[18F]fluorofolic acid displayed increased accumulation in atherosclerotic plaques through in vitro autoradiography. Specific binding of the radiotracer correlated with FR-ß-expressing macrophages. These results demonstrate high FR-ß expression in atherosclerotic lesions of human carotid tissue correlating with CD68-positive macrophages. Areas of high 3'-aza-2'-[18F]fluorofolic acid binding within the lesions represented FR-ß-expressing macrophages. Selectively targeting FR-ß-positive macrophages through folate-based radiopharmaceuticals may be useful for noninvasive imaging of plaque inflammation.

Synthesis and preliminary biological evaluation of O-2((2-[(18)F]fluoroethyl)methylamino)ethyltyrosine ([(18)F]FEMAET) as a potential cationic amino acid PET tracer for tumor imaging.

Amino acid transport is an attractive target for oncologic imaging. Despite a high demand of cancer cells for cationic amino acids, their potential as PET probes remains unexplored. Arginine, in particular, is involved in a number of biosynthetic pathways that significantly influence carcinogenesis and tumor biology. Cationic amino acids are transported by several cationic transport systems including, ATB(0,+) (SLC6A14), which is upregulated in certain human cancers including cervical, colorectal and estrogen receptor-positive breast cancer. In this work, we report the synthesis and preliminary biological evaluation of a new cationic analog of the clinically used PET tumor imaging agent O-(2-[(18)F]fluroethyl)-L-tyrosine ([(18)F]FET), namely O-2((2-[(18)F]fluoroethyl)methylamino)ethyltyrosine ([(18)F]FEMAET). Reference compound and precursor were prepared by multi-step approaches. Radiosynthesis was achieved by no-carrier-added nucleophilic [(18)F]fluorination in 16-20% decay-corrected yields with radiochemical purity >99%. The new tracer showed good stability in vitro and in vivo. Cell uptake assays demonstrated that FEMAET and [(18)F]FEMAET accumulate in prostate cancer (PC-3) and small cell lung cancer cells (NCI-H69), with an energy-dependent mechanism. Small animal PET imaging with NCI-H69 xenograft-bearing mice revealed good tumor visualization comparable to [(18)F]FET and low brain uptake, indicating negligible transport across the blood-brain barrier. In conclusion, the non-natural cationic amino acid PET probe [(18)F]FEMAET accumulates in cancer cells in vitro and in vivo with possible involvement of ATB(0,+).

Synthesis and biological evaluation of (18)F-labeled Fluoroethoxy tryptophan analogues as potential PET tumor imaging agents.

As a continuation of our research efforts toward the development of tryptophan-based radiotracers for tumor imaging with positron emission tomography (PET), three new fluoroethoxy tryptophan analogues were synthesized and evaluated in vivo. These new tracers (namely, 4-(2-[(18)F]fluoroethoxy)-dl-tryptophan ([(18)F]4-FEHTP), 6-(2-[(18)F]fluoroethoxy)-dl-tryptophan ([(18)F]6-FEHTP), and 7-(2-[(18)F]fluoroethoxy)-dl-tryptophan ([(18)F]7-FEHTP) carry the fluoroethoxy side chain either at positions 4-, 6-, or 7- of the indole core. Reference compounds and precursors were synthesized by multistep approaches. Radiosynthesis was accomplished by no-carrier-added nucleophilic (18)F-fluorination following either an indirect approach (O-alkylation of the corresponding hydroxytryptophan with [(18)F]fluoroethyltosylate) or a direct approach (nucleophilic [(18)F] fluorination using a protected mesyl precursor). Radiochemical yields (decay corrected) for both methods were in the range of 10-18%. Small animal PET imaging with xenograft-bearing mice revealed the highest tumor/background ratio for [(18)F]6-FEHTP which, in a direct comparison, outperformed the other two tryptophan tracers and also the well-established tyrosine analogue O-(2-[(18)F]fluoroethyl)-l-tyrosine ([(18)F]l-FET). Investigation of the transport mechanism of [(18)F]6-FEHTP in small cell lung cancer cells (NCI-H69) revealed that it is most probably taken up exclusively via the large neutral amino acid transporter(s) (LAT).

In vitro and in vivo evaluation of N-{2-[4-(3-Cyanopyridin-2-yl)piperazin-1-yl]ethyl}-3-[(11) C]methoxybenz-amide, a positron emission tomography (PET) radioligand for dopamine D4 receptors, in rodents.

The D4 dopamine receptor belongs to the D2 -like family of dopamine receptors, and its exact regional distribution in the central nervous system is still a matter of considerable debate. The availability of a selective radioligand for the D4 receptor with suitable properties for positron emission tomography (PET) would help resolve issues of D4 receptor localization in the brain, and the presumed diurnal change of expressed protein in the eye and pineal gland. We report here on in vitro and in vivo characteristics of the high-affinity D4 receptor-selective ligand N-{2-[4-(3-cyanopyridin-2-yl)piperazin-1-yl]ethyl}-3-[(11) C]methoxybenzamide ([(11) C]2) in rat. The results provide new insights on the in vitro properties that a brain PET dopamine D4 radioligand should possess in order to have improved in vivo utility in rodents.

Development and evaluation of novel PET tracers for imaging cannabinoid receptor type 2 in brain.

The cannabinoid receptor type 2 (CB2) has a very low expression level in brain tissue under basal conditions, but it is up-regulated in diverse pathological conditions. Two promising lead structures from the literature, N-((3S,5S,7S)-adamantan-1-yl)-8-methoxy-4-oxo-1-pentyl-1,4-dihydroquinoline-3-carboxamide and 8-butoxy-N-(2-fluoro-2-phenylethyl)-7-methoxy-2-oxo-1,2-dihydroquinoline-3-carboxamide - designated KD2 and KP23, respectively - were evaluated as potential PET ligands for imaging CB2. Both KD2 and KP23 were synthesized and labeled with carbon-11. In vitro autoradiographic studies on rodent spleen tissues showed that [(11)C]KD2 exhibits superior properties. A pilot study using [(11)C]KD2 on human post mortem ALS spinal cord slices indicated high CB2 expression level and specific binding, a very exciting finding if considering the future diagnostic application of CB2 ligands and their utility in therapy monitoring. In vivo blocking studies in rats with [(11)C]KD2 showed also high specific uptake in spleen tissue. Although the protein-bound fraction is relatively high, KD2 or KD2 derivatives could be very useful tools for the non-invasive investigation of CB2 levels under various neuroinflammatory conditions.

Investigating group formation: An experiment on the distribution of extraversion in educational settings.

Group formation plays a crucial role in enhancing collaborative learning experiences. This study investigates the impact of extraversion as a criterion for group formation on collaborative learning outcomes. A total of 180 students participated in the experiment and were assigned to groups that were homogeneously or heterogeneously distributed in terms of extraversion. The groups met weekly and worked on group assignments throughout the semesters. The first hypothesis posed the outcomes to be explainable at the group-level. Surprisingly, the results show that groups with a homogeneous distribution of extraversion reported higher levels of group work satisfaction than those with a heterogeneous distribution, in contrast to the second hypothesis and the group hierarchy theory. These findings emphasize the potential of considering personality traits when forming groups and extend the existing literature on group formation. The study takes a critical stance by addressing normative definitions of leadership. Future research is suggested to further enhance collaborative learning experiences using similar interdisciplinary and experimental methods.

Radiolabeling and in vitro /in vivo evaluation of N-(1-adamantyl)-8-methoxy-4-oxo-1-phenyl-1,4-dihydroquinoline-3-carboxamide as a PET probe for imaging cannabinoid type 2 receptor.

The cannabinoid type 2 (CB2) receptor plays an important role in neuroinflammatory and neurodegenerative diseases such as multiple sclerosis, amyotrophic lateral sclerosis, and Alzheimer's disease and is therefore a very promising target for therapeutic approaches as well as for imaging. Based on the literature, we identified one 4-oxoquinoline derivative(designated KD2) as the lead structure. It was synthesized, radiolabeled and evaluated as a potential imaging tracer for CB2. [11C]KD2 was obtained in 99% radiochemical purity.Moderate blood­brain barrier (BBB) passage was predicted for KD2 from an in vitro transport assay with P-glycoprotein-transfected Madin Darby canine kidney cells. No efflux of KD2 by P-glycoprotein was detected. In vitro autoradiography of rat and mouse spleen slices demonstrated that [11C]KD2 exhibits high specific binding towards CB2. High spleen uptake of [11C]KD2 was observed in dynamic positron emission tomography(PET) studies with Wistar rats and its specificity was confirmed by displacement study with a selective CB2 agonist, GW405833. A pilot autoradiography study with post-mortem spinal cord slices from amyotrophic lateral sclerosis (ALS)patients with [11C]KD2 suggested the presence of CB2 receptors under disease conditions. Specificity of [11C]KD2 binding could also be demonstrated on these human tissues. In conclusion, [11C]KD2 shows good in vitro and in vivo properties as a potential PET tracer for CB2.

Radiosynthesis and preclinical evaluation of 3'-Aza-2'-[(18)F]fluorofolic acid: a novel PET radiotracer for folate receptor targeting.

The folate receptor (FR) has been identified as a valuable target for the imaging of cancer and activated macrophages, involved in inflammatory and autoimmune diseases via positron emission tomography (PET). Therefore, conjugates of folic acid have been synthesized by coupling of a radiolabeled prosthetic group to the glutamate part of folic acid (pendent approach). In this work, we present a novel class of folates, where the phenyl ring of folic acid was isosterically replaced by a pyridine moiety for direct labeling with [(18)F]fluoride (integrated approach). 3'-Azafolic acid and its 2'-halogenated derivatives (2'-chloro and 2'-fluoro) were evaluated in vitro to determine their binding affinity. 3'-Aza-2'-[(18)F]fluorofolic acid ([(18)F]6) was obtained, starting from N(2)-acetyl-3'-aza-2'-chlorofolic acid di-tert-butylester (2), in a maximum decay corrected radiochemical yield of about 9% in ≥98% radiochemical purity and high specific activities of 35-127 GBq/µmol. Binding affinity to the FR was high (IC(50) = 0.8 ± 0.2 nM), and the radiotracer was stable in human plasma over 4 h at 37 °C. No degradation or defluorination was detected after incubation of the radiotracer for 1 h at 37 °C with human and murine liver microsomes and human S9-fraction. In vivo PET imaging and biodistribution studies with mice demonstrated a high and specific uptake in FR-positive KB tumor xenografts (12.59 ± 1.77% ID/g, 90 min p.i.). A high and specific accumulation of radioactivity was observed in the kidneys (57.33 ± 8.40% ID/g, 90 min p.i.) and salivary glands (14.09 ± 0.93% ID/g, 90 min p.i.), which are known to express the FR and nonspecific uptake found in the liver (10.31 ± 2.37% ID/g, 90 min p.i.). Preinjection of folic acid resulted in a >85% reduced uptake of [(18)F]6 in FR-positive tissues (xenografts, kidneys, and salivary glands). Furthermore, no radioactive metabolites were detected in the blood, urine, or tumor tissue, 30 min p.i. These characteristics indicate that this new (18)F-labeled 3'-azafolate is an appropriate tool for imaging FR-positive (malignant) tissue.

Synthesis and biological evaluation of a new acyclic pyrimidine derivative as a probe for imaging herpes simplex virus type 1 thymidine kinase gene expression.

With the idea of finding a more selective radiotracer for imaging herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene expression by means of positron emission tomography (PET), a novel [¹8F]fluorine radiolabeled pyrimidine with 4-hydroxy-3-(hydroxymethyl)butyl side chain at N-1 (HHB-5-[¹8F]FEP) was prepared and evaluated as a potential PET probe. Unlabeled reference compound, HHB-5-FEP, was synthesized via a five-step reaction sequence starting from 5-(2-acetoxyethyl)-4-methoxypyrimidin-2-one. The radiosynthesis of HHB-[¹8F]-FEP was accomplished by nucleophilic radiofluorination of a tosylate precursor using [¹8F]fluoride-cryptate complex in 45% ± 4 (n = 4) radiochemical yields and high purity (>99%). The biological evaluation indicated the feasibility of using HHB-5-[¹8F]FEP as a PET radiotracer for monitoring HSV1-tk expression in vivo.

[18F]fluoro-deoxy-glucose folate: a novel PET radiotracer with improved in vivo properties for folate receptor targeting.

The folate receptor (FR) is upregulated in various cancer types (FR-α isoform) and in activated macrophages (FR-ß isoform) which are involved in inflammatory and autoimmune diseases, but its expression in healthy tissues and organs is highly restricted to only a few sites (e.g kidneys). Therefore, the FR is a promising target for imaging and therapy of cancer and inflammation using folate-based radiopharmaceuticals. Herein, we report the synthesis and evaluation of a novel folic acid conjugate with improved properties suitable for positron emission tomography (PET). [(18)F]-fluoro-deoxy-glucose folate ([(18)F]3) was synthesized based on the click chemistry approach using 2-deoxy-2-[(18)F]fluoroglucopyranosyl azide and a folate alkyne derivative. The novel radiotracer [(18)F]3 was produced in good radiochemical yields (25% d.c.) and high specific radioactivity (90 GBq/µmol). Compared to previously published (18)F-folic acid derivatives, an increase in hydrophilicity was achieved by using a glucose entity as a prosthetic group. Biodistribution and PET imaging studies in KB tumor-bearing mice showed a high and specific uptake of the radiotracer in FR-positive tumors (10.03 ± 1.12%ID/g, 60 min p.i.) and kidneys (42.94 ± 2.04%ID/g, 60 min p.i.). FR-unspecific accumulation of radioactivity was only found in the liver (9.49 ± 1.13%ID/g, 60 min p.i.) and gallbladder (17.59 ± 7.22%ID/g, 60 min p.i.). No radiometabolites were detected in blood, urine, and liver tissue up to 30 min after injection of [(18)F]3. [(18)F]-fluoro-deoxy-glucose-folate ([(18)F]3) is thus a promising PET radioligand for imaging FR-positive tumors.

Interactions between phytochemical components of Sutherlandia frutescens and the antiretroviral, atazanavir in vitro: implications for absorption and metabolism.

PURPOSE: African traditional medicinal plants, such as Sutherlandia frutescens have the potential to interact pharmacokinetically with the protease inhibitor class of antiretrovirals, thereby impacting on their safety and efficacy. The effects of extracts and phytochemical components of Sutherlandia frutescens, on the in vitro absorption and metabolism of the protease inhibitor, atazanavir were thus investigated. METHODS: Aqueous and methanolic extracts of Sutherlandia frutescens were prepared by freeze-drying of hot water and methanol decoctions of Sutherlandia frutescens plant material respectively, whilst crude triterpenoid glycoside and flavonol glycoside fractions were isolated by solvent extraction and subsequent column chromatography. Atazanavir was quantitated in the absence or presence of these compounds as well as commercially available purported constituents of Sutherlandia frutescens, namely, L-canavanine, L-GABA and D-pinitol, after a one hour co-incubation in Caco-2 cell monolayers and human liver microsomes. RESULTS: The triterpenoid and flavonol glycoside fractions were found to be present in the aqueous and methanolic extracts of Sutherlandia frutescens and were shown to contain the sutherlandiosides and sutherlandins known to be present in Sutherlandia frutescens. The aqueous extract and D-pinitol significantly reduced atazanavir accumulation by Caco-2 cells, implying a decrease in atazanavir absorption, whilst the opposite was true for the triterpenoid glycoside fraction. Both the aqueous and methanolic extracts inhibited atazanavir metabolism in human liver microsomes, whilst enhanced atazanavir metabolism was exhibited by the triterpenoid glycoside fraction. CONCLUSIONS: The extracts and phytochemical components of Sutherlandia frutescens influenced the accumulation of atazanavir by Caco-2 cells and also affected ATV metabolism in human liver microsomes. These interactions may have important implications on the absorption and metabolism and thus the overall oral bioavailability of atazanavir.

Potential pharmacokinetic interactions between antiretrovirals and medicinal plants used as complementary and African traditional medicines.

The use of traditional/complementary/alternate medicines (TCAMs) in HIV/AIDS patients who reside in Southern Africa is quite common. Those who use TCAMs in addition to antiretroviral (ARV) treatment may be at risk of experiencing clinically significant pharmacokinetic (PK) interactions, particularly between the TCAMs and the protease inhibitors (PIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). Mechanisms of PK interactions include alterations to the normal functioning of drug efflux transporters, such as P-gp and/or CYP isoenzymes, such a CYP3A4 that mediate the absorption and elimination of drugs in the small intestine and liver. Specific mechanisms include inhibition and activation of these proteins and induction via the pregnane X receptor (PXR). Several clinical studies and case reports involving ARV-herb PK interactions have been reported. St John's Wort, Garlic and Cat's Claw exhibited potentially significant interactions, each with a PI or NNRTI. The potential for these herbs to induce PK interactions with drugs was first identified in reports of in vitro studies. Other in vitro studies have shown that several African traditional medicinal (ATM) plants and extracts may also demonstrate PK interactions with ARVs, through effects on CYP3A4, P-gp and PXR. The most complex effects were exhibited by Hypoxis hemerocallidea, Sutherlandia frutescens, Cyphostemma hildebrandtii, Acacia nilotica, Agauria salicifolia and Elaeodendron buchananii. Despite a high incidence of HIV/AIDs in the African region, only one clinical study, between efavirenz and Hypoxis hemerocallidea has been conducted. However, several issues/concerns still remain to be addressed and thus more studies on ATMs are warranted in order for more meaningful data to be generated and the true potential for such interactions to be determined.

Neuroprotective and axon growth-promoting effects following inflammatory stimulation on mature retinal ganglion cells in mice depend on ciliary neurotrophic factor and leukemia inhibitory factor.

After optic nerve injury retinal ganglion cells (RGCs) normally fail to regenerate axons in the optic nerve and undergo apoptosis. However, lens injury (LI) or intravitreal application of zymosan switch RGCs into an active regenerative state, enabling these neurons to survive axotomy and to regenerate axons into the injured optic nerve. Several factors have been proposed to mediate the beneficial effects of LI. Here, we investigated the contribution of glial-derived ciliary neurotrophic factor (CNTF) to LI-mediated regeneration and neuroprotection using wild-type and CNTF-deficient mice. In wild-type mice, CNTF expression was strongly upregulated in retinal astrocytes, the JAK/STAT3 pathway was activated in RGCs, and RGCs were transformed into an active regenerative state after LI. Interestingly, retinal LIF expression was correlated with CNTF expression after LI. In CNTF-deficient mice, the neuroprotective and axon growth-promoting effects of LI were significantly reduced compared with wild-type animals, despite an observed compensatory upregulation of LIF expression in CNTF-deficient mice. The positive effects of LI and also zymosan were completely abolished in CNTF/LIF double knock-out mice, whereas LI-induced glial and macrophage activation was not compromised. In culture CNTF and LIF markedly stimulated neurite outgrowth of mature RGCs. These data confirm a key role for CNTF in directly mediating the neuroprotective and axon regenerative effects of inflammatory stimulation in the eye and identify LIF as an additional contributing factor.

Exogenous CNTF stimulates axon regeneration of retinal ganglion cells partially via endogenous CNTF.

Intravitreal injections of exogenous CNTF stimulate axon regeneration of RGCs in vivo. Nevertheless, controversy exists over the ability of exogenous CNTF to directly stimulate axon regeneration of mature RGCs. Here we demonstrate that CNTF potently stimulated axon outgrowth of mature RGCs in culture in a JAK/STAT3- and PI3K/AKT-signaling pathway-dependent fashion and stronger than oncomodulin. Additional cAMP elevation or inhibition of MAPK activity increased these effects. In vivo intravitreal injections of exogenous CNTF induced endogenous CNTF expression in astrocytes in a manner that depended on the MAPK/ERK-signaling pathway activation. Reduction of endogenous CNTF expression by MAPK/ERK pathway inhibitors or its absence in CNTF deficient mice markedly reduced the neurite growth-promoting effects of exogenous CNTF. These data demonstrate that CNTF is a potent axon growth-promoting factor for mature RGCs. However, exogenously applied CNTF stimulates RGCs in vivo partially indirectly via a mechanism that depends on astrocyte-derived CNTF.

Crystallins of the beta/gamma-superfamily mimic the effects of lens injury and promote axon regeneration.

Adult retinal ganglion cells (RGCs) can survive axotomy and regrow lengthy axons when exposed to lens injury (LI). The neuroprotective and axon-growth-promoting effects of LI have been attributed to an infiltration of activated macrophages into the inner eye and recently also to astrocyte-derived CNTF. The present work reveals that certain purified lens proteins (crystallins) cause the effects of LI. Intravitreal injections of beta- or gamma-crystallins, but not of alpha-crystallin, strongly enhanced axon regeneration from retinal explants in culture, within peripheral nerve grafts or the crushed optic nerve. Deposition of the effective crystallins within the vitreous body was also associated with an influx of circulating macrophages and an activation of retinal astrocytes, Müller cells, and resident microglia. Furthermore beta-crystallin induced CNTF expression in retinal astrocytes and activation of CNTF's major downstream signaling pathway (JAK/STAT3) when intravitreally injected or added to the culture medium ex vivo. Consistently, in culture the addition of beta- and gamma-crystallins to the medium also increased axon regeneration from retinal explants. These results demonstrate that crystallins of the beta/gamma-superfamily are the lens-derived activators of cascades, which lead to axonal regeneration and suggest that their effects might be mediated by astrocyte-derived CNTF.

Astrocyte-derived CNTF switches mature RGCs to a regenerative state following inflammatory stimulation.

Retinal ganglion cells (RGCs) normally fail to regenerate injured axons and undergo apoptosis soon after injury. We have recently shown that lens injury (LI) or intravitreally applied zymosan allow RGCs to survive axotomy and regenerate axons in the injured optic nerve. Activated macrophages and oncomodulin have been suggested to be the principal mediators of this phenomenon. However, several lines of evidence show that macrophage-derived factors alone cannot account for all the beneficial effects of intraocular inflammation. We show here that LI or zymosan induce upregulation of ciliary neurotrophic factor (CNTF) in retinal astrocytes and release CNTF independent of macrophages and activate the transcription factor signal transducers and activators of transcription 3 (STAT3) in RGCs. Levels of CNTF expressed in retinal glia and STAT3 activation in RGC were correlated with the time course of RGCs switching to an active regenerative state. Intravitreal injections of antibodies against CNTF or a Janus-kinase inhibitor compromised the beneficial effects of LI, whereas an antiserum against oncomodulin was ineffective. Like the action of CNTF, the effects of LI were potentiated by drugs that increase intracellular cAMP levels, resulting in strong axon regeneration in vivo. These data indicate that astrocyte-derived CNTF is a major contributor to the neuroprotective and axon-growth-promoting effects of LI and zymosan. These findings could lead to the development of a therapeutic principle for promoting axon regeneration in the CNS as a whole.

Non-steroidal anti-inflammatory agents, tolmetin and sulindac attenuate quinolinic acid (QA)-induced oxidative stress in primary hippocampal neurons and reduce QA-induced spatial reference memory deficits in male Wistar rats.

Accumulating evidence suggests that anti-inflammatory agents and antioxidants have neuroprotective properties and may be beneficial in the treatment of neurodegenerative disorders. In the present study, the possible neuroprotective properties of tolmetin and sulindac were investigated using quinolinic acid (QA)-induced neurotoxicity as well as behavioral studies. QA, a metabolite of the tryptophan-kynurenine pathway, significantly induces lipid peroxidation, superoxide anion generation and decreases cell viability in primary hippocampal neurons established from one day old rat pups. However, co-incubation of the neurons with tolmetin or sulindac markedly reduces oxidative stress and enhances cell viability. Animals were trained in a Morris water maze for four consecutive days and thereafter received 0.6 micromol of QA intrahippocampally. The animals were divided into groups and were treated with either tolmetin or sulindac (5 mg/kg twice a day for five days). During test trials, the time taken for each rat to find the submerged platform was recorded over a period of two weeks. Animals were thereafter sacrificed and the hippocampi analyzed for protein carbonyl and glutathione content. The results show that both sulindac and tolmetin reduce the QA-induced spatial memory deficit and sulindac treated animals respond better in the water maze compared to the tolmetin treated animals. Both agents also reduce protein oxidation in rat hippocampus and attenuate the decrease in hippocampal glutathione content induced by QA. This study indicates that the antioxidant properties of tolmetin and sulindac may be beneficial in the treatment of neurodegenerative disorders such as Alzheimer's disease.

Mechanisms by which acyclovir reduces the oxidative neurotoxicity and biosynthesis of quinolinic acid.

The concentration of the endogenous neurotoxin quinolinic acid (QA) is increased in the central nervous system of mice with herpes simplex encephalitis. We have previously shown that the antiherpetic agent acyclovir (AC) has the ability to reduce QA-induced neuronal damage in rat brain, by attenuating lipid peroxidation. The mechanism by which QA induces lipid peroxidation includes the enhancement of the iron (Fe)-mediated Fenton reaction and the generation of free radicals, such as the superoxide anion (O(2)(-)). Thus, the present study determined whether AC has the ability to reduce Fe(2+)-induced lipid peroxidation, O(2)(-) generation and QA-induced superoxide anion generation, and to bind free Fe. O(2)(-) and Fe(2+) are also cofactors of the enzymes, indoleamine-2,3-dioxygenase (IDO) and 3-hydroxyanthranilate-3,4-dioxygenase (3-HAO) respectively. These enzymes catalyse steps in the biosynthesis of QA; thus, the effect of AC on their activity was also investigated. AC significantly attenuates Fe(2+)-induced lipid peroxidation and O(2)(-) generation. AC reduces O(2)(-) generation in the presence of QA and strongly binds Fe(2+) and Fe(3+). It also reduces the activity of both IDO and 3-HAO, which could be attributed to the superoxide anion scavenging and iron binding properties, respectively, of this drug.

RNA interference suggests sulfakinins as satiety effectors in the cricket Gryllus bimaculatus.

In the Mediterranean field cricket, Gryllus bimaculatus, the action of sulfakinin (SK) gene expression on food intake, food transport in the gut and carbohydrate digestion (alpha-amylase activity) was investigated by using the RNA interference (RNAi) method. Injection of SK double-stranded (ds) RNA into the abdomen of female adults and last instar larvae led to a systemic silencing of the SK gene, as was shown by RT-PCR studies. In adults, suppression of SK gene expression was effective from the first day after injection up to at least the third day. Treatment of the adult crickets by injection or feeding of dsRNA led to a stimulation of the food intake. Assuming that the gene silencing is followed by a depletion of the SK in tissues and/or haemolymph implies an inhibitiory role of the native SK peptides on food intake. The alpha-amylase activity in vitro in the midgut tissue and in the secretions of adult females was not affected by silencing the SK gene.