Quantification of dopamine transporter binding using [18F]FP-beta-CIT and positron emission tomography.

The purpose of this study was to compare different kinetic and semi-quantitative methods for analysing human [18F]FP-beta-CIT studies: plasma input models, simplified (SRTM) and full (FRTM) reference tissue models, standard uptake values (SUV) and SUV ratios (SUVr). Both simulations and clinical evaluations were performed to determine the effects of noise, scan duration and blood volume on Akaike model selection, and on precision and accuracy of estimated parameters. For typical noise levels (COV approximately 2.5%) and scan durations (<90 mins), simulations provided poor fits (Akaike criterion) in case of reversible plasma input models showing a relatively high number of outliers compared with the two-tissue irreversible model. Reference tissue models provided more reliable fits, which were nearly independent of noise and scan duration. For clinical data, two tissue irreversible and reversible plasma input models fitted striatum curves equally well (Akaike criterion). BP with plasma input models were less precise and contained more outliers than BP obtained with SRTM or FRTM. Among all methods tested, SRTM showed the highest contrast between patients and controls. When differentiating between patients and controls, SUVr performed almost equally well as SRTM, although contrast between striatum and background was lower. In conclusion, SRTM provided BP estimates with the highest precision and accuracy. Moreover, SRTM provided good contrast between patients and controls, and between striatum and background. SRTM is therefore the method of choice for quantitative [18F]FP-beta-CIT studies. SUVr might be an alternative for larger clinical trials.     

Intradermal capsaicin causes dose-dependent pain, allodynia, and hyperalgesia in humans.

BACKGROUND: Intradermal capsaicin is a human pain model that produces reliable pain and sensitization. This model facilitates controlled testing of analgesic efficacy via a crossover design while minimizing confounding variables in clinical pain states and retaining sufficient power with small samples. METHODS: To determine the lowest dose of capsaicin that produces consistent neurosensory measures, we administered 0.1, 1, 10, or 100 microg to healthy volunteers in a blinded manner (N = 19). Pain scores were recorded at 0, 5, 10, 15, and 60 minutes on a visual analog scale from 0 to 100. Areas and intensities of mechanical allodynia (foam brush stimulus) and pinprick hyperalgesia (von Frey test) were quantified at 15 and 60 minutes, as were flare areas. RESULTS: Capsaicin produced dose-dependent increases in spontaneous pain (p = .013), the area and intensity of mechanical allodynia (p = .006 and p < .001, respectively), the area and intensity of pinprick hyperalgesia (p = .010 and p = .014, respectively), and the flare area (p = .010). The 10 microg dose produced greater spontaneous pain than the 1 microg dose (p = .017). The 100 microg dose produced greater spontaneous pain than the 10 microg, but the difference was not statistically significant. CONCLUSION: The 10 and 100 microg capsaicin doses produced robust pain measures across a range of modalities, and lower doses produced minimal effects. Whereas most studies use 100 microg, using a lower dose is reasonable and may facilitate detection of subtle analgesic effects--particularly with nonopioid analgesics--and drugs can be tested in lower doses, minimizing adverse side effects.     

Optical Pretargeting of Tumor with Fluorescent MORF Oligomers

Objective Pretargeting with radioactivity has significantly improved tumor to normal tissue radioactivity ratios over conventional antibody imaging in both animal studies and clinical trials. This laboratory is investigating DNA analogues such as phosphorodiamidate morpholinos (MORFs) for pretargeting using technetium-99m (^99mTc) for detection. However, the unique properties of fluorescence activation and quenching combined with oligomers with their unique properties of hybridization may be particularly useful when used together for pretargeting with optical detection. The use of linear fluorophore-conjugated oligomer duplexes have been little used in animals, and to our knowledge, have not previously been considered for pretargeting applications. Methods A MORF/cDNA pair was selected such that when hybridized, the fluorescence of the Cy5.5-conjugated 25 mer MORF (Cy5.5–MORF25) is inhibited with a BHQ3-conjugated 18 mer complementary DNA (BHQ3–cDNA18). The short BHQ3–cDNA18 was selected to dissociate in the presence of a long cMORF25 in the pretargeted tumor, thus releasing the inhibitor from the Cy5.5 emitter. In this manner, the Cy5.5 fluorescence will be inhibited everywhere but in the target. The dissociation was first examined in vitro by adding the duplex to the cMORF25 both in solution and immobilized on polystyrene microspheres and by surface plasmon resonance (SPR). Thereafter, biotinylated cMORF25 immobilized on streptavidin polystyrene microspheres were administered intramuscularly in one thigh of hairless SKH-1 mice as target while an identical weight of the identical microspheres but without the cMORF25 was administered in the contralateral thigh as control. The animals then received IV the Cy5.5–MORF25/BHQ3–cDNA18 duplex or equal molar dosage of single-chain Cy5.5–MORF25 and were imaged. Results The SPR studies showed that the immobilized cDNA18 rapidly captured the flowing MORF25 to provide a duplex with a slow dissociation rate constant. Furthermore, when cMORF25 was next allowed to flow over the now immobilized duplex, the cDNA18 was unable to prevent dissociation of the heteroduplex and the formation and release of the cMORF25-MORF25 homoduplex. Images of animals obtained soon after receiving the Cy5.5–MORF25 singlet showed intense whole body fluorescence obscuring the target thigh. However, only 5 minutes after receiving the Cy5.5–MORF25/BHQ3–cDNA18 duplex, the target thigh was clearly visible along with only the kidneys. Conclusions This first study of optical pretargeting provides a proof of concept that oligomer pretargeting found to be useful with radioactivity detection is applicable with fluorescent detection as well. In addition, our results demonstrate that by using linear oligomers for optical pretargeting, chain lengths (and base sequences) may be manipulated to provide duplexes with stabilities and fluorescence inhibition optimized for pretargeting and other in vivo applications of optical imaging.     

Endogenous kynurenic acid disrupts prepulse inhibition.

BACKGROUND: Recent studies show that endogenous levels of kynurenic acid (KYNA) are increased in the cerebrospinal fluid of schizophrenic patients. Prepulse inhibition (PPI) of the acoustic startle reflex is an operational measure of sensorimotor gating that is reduced in neuropsychiatric disorders, such as schizophrenia. Previous studies show that administration of N-methyl-D-aspartate (NMDA) receptor antagonists, such as phencyclidine or MK-801, leads to deficits in sensorimotor gating that mimic those observed in schizophrenic patients. METHODS: The present study examined the effects of the endogenous NMDA receptor antagonist KYNA on startle and PPI in rats. Elevation of endogenous brain levels of KYNA was achieved through intraperitoneal (IP) administration of kynurenine (100 mg/kg), the precursor of KYNA, or by intravenous administration of PNU 156561A (10 mg/kg). RESULTS: A fourfold increase in brain KYNA levels, as induced by kynurenine or PNU 156561A, significantly reduced PPI. There were no differences in startle magnitudes between control rats and drug-treated rats. The disruption of PPI was restored by administration of the antipsychotic drugs haloperidol (.2 mg/kg, IP) or clozapine (7.5 mg/kg, IP). CONCLUSIONS: The present results suggest that brain KYNA serves as an endogenous modulator of PPI and are consistent with the hypothesis that KYNA contributes to the pathophysiology of schizophrenia.     

Interleukin 6 Influences Germinal Center Development and Antibody Production via a Contribution of C3 Complement Component

Mice rendered deficient for interleukin (IL) 6 by gene targeting were evaluated for their response to T cell–dependent antigens. Antigen-specific immunoglobulin (Ig)M levels were unaffected whereas all IgG isotypes showed varying degrees of alteration. Germinal center reactions occurred but remained physically smaller in comparison to those in the wild-type mice. This concurred with the observations that molecules involved in initial signaling events leading to germinal center formation were not altered (e.g., B7.2, CD40 and tumor necrosis factor R1). T cell priming was not impaired nor was a gross imbalance of T helper cell (Th) 1 versus Th2 cytokines observed. However, B7.1 molecules, absent from wild-type counterparts, were detected on germinal center B cells isolated from the deficient mice suggesting a modification of costimulatory signaling. A second alteration involved impaired de novo synthesis of C3 both in serum and germinal center cells from IL-6–deficient mice. Indeed, C3 provided an essential stimulatory signal for wild-type germinal center cells as both monoclonal antibodies that interrupted C3-CD21 interactions and sheep anti–mouse C3 antibodies caused a significant decrease in antigen-specific antibody production. In addition, germinal center cells isolated from C3–deficient mice produced a similar defect in isotype production. Low density cells with dendritic morphology were the local source of IL-6 and not the germinal center lymphocytes. Adding IL-6 in vitro to IL-6–deficient germinal center cells stimulated cell cycle progression and increased levels of antibody production. These findings reveal that the germinal center produces and uses molecules of the innate immune system, evolutionarily pirating them in order to optimally generate high affinity antibody responses.     

Combining Free Flap Reconstruction and Craniofacial Prosthetic Technique for Orbit,Scalp, and Temporal Defects

Objectives: To identify factors leading to successful application of prosthetic techniques following free flap reconstruction of the orbit, scalp, and temporal region. Study Design: Retrospective review. Methods: Twenty-eight patients who underwent free flap reconstruction for defects of these regions between 1989 and 1996 were reviewed for clinical parameters, flap loss, patient survival, and implant loss rate. Prosthetic usage rates were compared before and after introduction of a site-specific reconstructive algorithm. Results: Free flap success rate was 93%, whereas osseointegrated implant loss rate was 11%. In addition to implants, a reconstructive strategy that provided thin, vascular tissue between bone and skin, a flat platform in the temporal region, and preservation of orbital cavity depth led to increased prosthetic usage. Conclusions: Craniofacial prosthetic techniques can significantly augment the results of free flap surgery for the orbit, scalp, and temporal region. Successful combination of these techniques requires a site-specific surgical approach.