Preclinical evaluation of PAC1 targeting with radiolabeled Maxadilan.

There is an ongoing search for new tracers to optimize imaging of beta cell-derived tumors (insulinomas). The PAC1 receptor, expressed by insulinomas, can be used for targeting of these tumors. Here, we investigated whether radiolabeled maxadilan could be used for insulinoma imaging. Maxadilan was C- or N-terminally conjugated with DTPA (termed maxadilan-DPTA or DTPA-maxadilan respectively). BALB/c nude mice bearing subcutaneous INS-1 tumors were injected with either In-111-labeled maxadilan-DTPA or In-111-DTPA-maxadilan. Biodistribution studies were carried out at 1, 2 and 4 hours after injection and SPECT/CT imaging 1 and 4 hours after injection of maxadilan-DTPA-111In. Radiolabeling of maxadilan-DTPA (680 MBq/nmol) was more efficient than of DTPA-maxadilan (55 MBq/nmol). Conjugation with DTPA slightly reduced receptor binding affinity in vitro: IC50 values were 3.2, 21.0 and 21.0 nM for maxadilan, natIn-DTPA-maxadilan and maxadilan-DTPA-natIn respectively. Upon i.v. injection maxadilan-DTPA-111In accumulated specifically in INS-1 tumors (7.30 ± 1.87%ID/g) and in the pancreas (3.82 ± 0.22%ID/g). INS-1 tumors were clearly visualized by small animal SPECT/CT. In conclusion, this study showed that the high affinity of maxadilan to the PAC1 receptor was maintained after DTPA conjugation. Furthermore, radiolabeled maxadilan-DTPA accumulated specifically in INS-1 tumors and, therefore, may qualify as a useful tracer to image insulinomas.

Intraperitoneal oxidative stress in rabbits with papillomavirus-associated head and neck cancer induces tumoricidal immune response that is adoptively transferable.

PURPOSE: How tumors evade or suppress immune surveillance is a key question in cancer research, and overcoming immune escape is a major goal for lengthening remission after cancer treatment. Here, we used the papillomavirus-associated rabbit auricular VX2 carcinoma, a model for studying human head and neck cancer, to reveal the mechanisms underlying the antitumorigenic effects of intraperitoneal oxidative stress following O3/O2-pneumoperitoneum (O3/O2-PP) treatment. EXPERIMENTAL DESIGN: Solid auricular VX2 tumors were induced in immune-competent adult New Zealand White Rabbits. Animals were O3/O2-PP- or sham-treated, after which they underwent tumor ablation upon reaching no-go criteria. CD3(+) tumor-infiltrating lymphocytes (TIL) were evaluated by immunohistochemistry, and expression levels of 84 immune response genes were measured by quantitative real-time PCR. Adoptive transfer of peripheral blood leukocytes (PBL)-derived from animals with tumor regression-into control animals with progressing tumors was implemented to assess acquired tumor resistance functionally. RESULTS: Auricular VX2 tumors regressing after O3/O2-PP treatment exhibited increased levels of CD3(+) TILs; they also exhibited enhanced expression of genes that encode receptors involved in pattern recognition, molecules that are required for antigen presentation and T cell activation, and inflammatory mediators. Adoptive cell transfer of PBLs from donor rabbits with regressing tumors to recipient rabbits with newly implanted VX2 carcinoma resulted in acquired tumor resistance of the host and tumor regression. CONCLUSION: Intraperitoneal oxidative stress effectively converts the immune response against the papillomavirus-associated rabbit VX2 carcinoma from tumor permissive to tumoricidal and leads to a sustainable, adoptively transferable oncolytic immune response.

Treatment with ozone/oxygen-pneumoperitoneum results in complete remission of rabbit squamous cell carcinomas.

Head and neck squamous cell carcinomas (HNSCC) represent a group of metastasizing tumors with a high mortality rate in man and animals. Since the biomolecule ozone was found to inhibit growth of various carcinoma cells in vitro we here applied the highly aggressive and lethal VX2 carcinoma HNSCC tumor model of the New Zealand White rabbit to test whether ozone exerts antitumorous effects in vivo. Therapeutic insufflation of medical ozone/oxygen (O(3)/O(2)) gas mixture into the peritoneum (O(3)/O(2)-pneumoperitoneum) at an advanced stage of tumor disease led to a survival rate of 7/14 rabbits. Six of the seven surviving rabbits presented full tumor regression and the absence of local or distant lung metastases. Insufflation of pure oxygen (O(2)) resulted in a survival rate of 3/13 animals accompanied by full tumor remission in 2 of the 3 surviving animals. Of the 14 sham-treated animals only 1 had spontaneous tumor remission and survived. No adverse effects or changes in standard blood parameters were observed after repeated intraperitoneal insufflations of the O(3)/O(2) or O(2) gas. Animals with O(3)/O(2)-induced tumor eradication developed tolerance against reimplantation of the VX2 tumor. This could be reversed by immune suppression with a combination of dexamethasone and cyclosporin A suggesting an antitumorous effect of O(3)/O(2)-mediated activation of the body's own immunosurveillance. Although the exact mechanisms of action are still unclear the present data point to O(3)/O(2)-pneumoperitoneum as a promising new strategy in anticancer therapy.

Microglial activation and increased synthesis of complement component C1q precedes blood-brain barrier dysfunction in rats.

A reliable way to visualise the state of microglial activation is to monitor the microglial gene expression profile. Microglia are the only CNS resident cells that synthesise C1q, the recognition sub-component of the classical complement pathway, in vivo. C1q biosynthesis in resting ramified microglia is often low, but it increases dramatically in activated microglia. In this study, the expression of C1q was used to monitor microglial activation at all stages of 3-chloropropanediol-induced neurotoxicity, a new model of blood-brain barrier (BBB) breakdown. In rats, 3-chloropropanediol produces very focused lesions in the brain, characterised by early astrocyte swelling and loss, followed by neuronal death and barrier dysfunction. Using in situ hybridisation, immunohistochemistry, and real-time RT-PCR, we found that increased C1q biosynthesis and microglial activation precede BBB dysfunction by at least 18 and peak 48 h after injection of 3-chloropropanediol, which coincides with the onset of active haemorrhage. Microglial activation is biphasic; an early phase of global activation is followed by a later phase in which microglial activation becomes increasingly focused in the lesions. During the early phase, expression of the pro-inflammatory mediators interleukin-1beta (IL1beta), tumour necrosis factor alpha (TNFalpha) and early growth response-1 (Egr-1) increased in parallel with C1q, but was restricted to the lesions. Expression of C1q (but not IL1beta, TNFalpha or Egr-1) remains high after BBB function is restored, and is accompanied by late up-regulation of the C1q-associated serine proteases, C1r and C1s, suggesting that microglial biosynthesis of the activation complex of the classical pathway may support the removal of cell debris by activation of complement.

Functional properties and cellular distribution of the system A glutamine transporter SNAT1 support specialized roles in central neurons.

Glutamine, the preferred precursor for neurotransmitter glutamate and GABA, is likely to be the principal substrate for the neuronal System A transporter SNAT1 in vivo. We explored the functional properties of SNAT1 (the product of the rat Slc38a1 gene) by measuring radiotracer uptake and currents associated with SNAT1 expression in Xenopus oocytes and determined the neuronal-phenotypic and cellular distribution of SNAT1 by confocal laser-scanning microscopy alongside other markers. We found that SNAT1 mediates transport of small, neutral, aliphatic amino acids including glutamine (K0.5 approximately 0.3 mm), alanine, and the System A-specific analogue 2-(methylamino)isobutyrate. Amino acid transport is driven by the Na+ electrochemical gradient. The voltage-dependent binding of Na+ precedes that of the amino acid in a simultaneous transport mechanism. Li+ (but not H+) can substitute for Na+ but results in reduced Vmax. In the absence of amino acid, SNAT1 mediates Na+-dependent presteady-state currents (Qmax approximately 9 nC) and a nonsaturable cation leak with selectivity Na+, Li+ >> H+, K+. Simultaneous flux and current measurements indicate coupling stoichiometry of 1 Na+ per 1 amino acid. SNAT1 protein was detected in somata and proximal dendrites but not nerve terminals of glutamatergic and GABAergic neurons throughout the adult CNS. We did not detect SNAT1 expression in astrocytes but detected its expression on the luminal membranes of the ependyma. The functional properties and cellular distribution of SNAT1 support a primary role for SNAT1 in glutamine transport serving the glutamate/GABA-glutamine cycle in central neurons. Localization of SNAT1 to certain dopaminergic neurons of the substantia nigra and cholinergic motoneurons suggests that SNAT1 may play additional specialized roles, providing metabolic fuel (via alpha-ketoglutarate) or precursors (cysteine, glycine) for glutathione synthesis.

Molecular cloning and functional identification of mouse vesicular glutamate transporter 3 and its expression in subsets of novel excitatory neurons.

We have cloned and functionally characterized a third isoform of a vesicular glutamate transporter (VGLUT3) expressed on synaptic vesicles that identifies a distinct glutamatergic system in the brain that is partly and selectively promiscuous with cholinergic and serotoninergic transmission. Transport activity was specific for glutamate, was H(+)-dependent, was stimulated by Cl(-) ion, and was inhibited by Rose Bengal and trypan blue. Northern analysis revealed higher mRNA levels in early postnatal development than in adult brain. Restricted patterns of mRNA expression were observed in presumed interneurons in cortex and hippocampus, and projection systems were observed in the lateral and ventrolateral hypothalamic nuclei, limbic system, and brainstem. Double in situ hybridization histochemistry for vesicular acetylcholine transporter identified VGLUT3 neurons in the striatum as cholinergic interneurons, whereas VGLUT3 mRNA and protein were absent from all other cholinergic cell groups. In the brainstem VGLUT3 mRNA was concentrated in mesopontine raphé nuclei. VGLUT3 immunoreactivity was present throughout the brain in a diffuse system of thick and thin beaded varicose fibers much less abundant than, and strictly separated from, VGLUT1 or VGLUT2 synapses. Co-existence of VGLUT3 in VMAT2-positive and tyrosine hydroxylase -negative varicosities only in the cerebral cortex and hippocampus and in subsets of tryptophan hydroxylase-positive cell bodies and processes in differentiating primary raphé neurons in vitro indicates selective and target-specific expression of the glutamatergic/serotoninergic synaptic phenotype.

Identification of the differentiation-associated Na+/PI transporter as a novel vesicular glutamate transporter expressed in a distinct set of glutamatergic synapses.

Glutamate transport into synaptic vesicles is a prerequisite for its regulated neurosecretion. Here we functionally identify a second isoform of the vesicular glutamate transporter (VGLUT2) that was previously identified as a plasma membrane Na+-dependent inorganic phosphate transporter (differentiation-associated Na+/P(I) transporter). Studies using intracellular vesicles from transiently transfected PC12 cells indicate that uptake by VGLUT2 is highly selective for glutamate, is H+ dependent, and requires Cl- ion. Both the vesicular membrane potential (Deltapsi) and the proton gradient (DeltapH) are important driving forces for vesicular glutamate accumulation under physiological Cl- concentrations. Using an antibody specific for VGLUT2, we also find that this protein is enriched on synaptic vesicles and selective for a distinct class of glutamatergic nerve terminals. The pathway-specific, complementary expression of two different vesicular glutamate transporters suggests functional diversity in the regulation of vesicular release at excitatory synapses. Together, the two isoforms may account for the uptake of glutamate by synaptic vesicles from all central glutamatergic neurons.