Binding Affinity,Specificity and Comparative Biodistribution of the Parental Murine Monoclonal Antibody MX35 (Anti-NaPi2b) and Its Humanized Version Rebmab200

The aim of this preclinical study was to evaluate the characteristics of the monoclonal antibody Rebmab200, which is a humanized version of the ovarian-specific murine antibody MX35. This investigation contributes to the foundation for future clinical α-radioimmunotherapy of minimal residual ovarian cancer with ²¹¹At-Rebmab200. Here, the biodistribution of ²¹¹At-Rebmab200 was evaluated, as was the utility of ^99mTc-Rebmab200 for bioimaging. Rebmab200 was directly compared with its murine counterpart MX35 in terms of its in-vitro capacity for binding the immobilized NaPi2B epitope and live cells; we also assessed its biodistribution in nude mice carrying subcutaneous OVCAR-3 tumors. Tumor antigen and cell binding were similar between Rebmab200 and murine MX35, as was biodistribution, including normal tissue uptake and in-vivo tumor binding. We also demonstrated that ^99mTc-Rebmab200 can be used for single-photon emission computed tomography of subcutaneous ovarian carcinomas in tumor-bearing mice. Taken together, our data support the further development of Rebmab200 for radioimmunotherapy and diagnostics.     

Pregnenolone Sulfate Potentiates the Inwardly Rectifying K+ Channel Kir2.3

Background

Neurosteroids have various physiological and neuropsychopharmacological effects. In addition to the genomic effects of steroids, some neurosteroids modulate several neurotransmitter receptors and channels, such as N-methyl-D-aspartate receptors, γ-aminobutyric acid type A (GABA_A) receptors, and σ₁ receptors, and voltage-gated Ca²⁺ and K⁺ channels. However, the molecular mechanisms underlying the various effects of neurosteroids have not yet been sufficiently clarified. In the nervous system, inwardly rectifying K⁺ (Kir) channels also play important roles in the control of resting membrane potential, cellular excitability and K⁺ homeostasis. Among constitutively active Kir2 channels in a major Kir subfamily, Kir2.3 channels are expressed predominantly in the forebrain, a brain area related to cognition, memory, emotion, and neuropsychiatric disorders.

Methodology/Principal Findings

The present study examined the effects of various neurosteroids on Kir2.3 channels using the Xenopus oocyte expression assay. In oocytes injected with Kir2.3 mRNA, only pregnenolone sulfate (PREGS), among nine neurosteroids tested, reversibly potentiated Kir2.3 currents. The potentiation effect was concentration-dependent in the micromolar range, and the current-voltage relationship showed inward rectification. However, the potentiation effect of PREGS was not observed when PREGS was applied intracellularly and was not affected by extracellular pH conditions. Furthermore, although Kir1.1, Kir2.1, Kir2.2, and Kir3 channels were insensitive to PREGS, in oocytes injected with Kir2.1/Kir2.3 or Kir2.2/Kir2.3 mRNA, but not Kir2.1/Kir2.2 mRNA, PREGS potentiated Kir currents. These potentiation properties in the concentration-response relationships were less potent than for Kir2.3 channels, suggesting action of PREGS on Kir2.3-containing Kir2 heteromeric channels.

Conclusions/Significance

The present results suggest that PREGS acts as a positive modulator of Kir2.3 channels. Kir2.3 channel potentiation may provide novel insights into the various effects of PREGS.     

Thallium transport and the evaluation of olfactory nerve connectivity between the nasal cavity and olfactory bulb

Little is known regarding how alkali metal ions are transported in the olfactory nerve following their intranasal administration. In this study, we show that an alkali metal ion, thallium is transported in the olfactory nerve fibers to the olfactory bulb in mice. The olfactory nerve fibers of mice were transected on both sides of the body under anesthesia. A double tracer solution (thallium-201, (201)Tl; manganese-54, (54)Mn) was administered into the nasal cavity the following day. Radioactivity in the olfactory bulb and nasal turbinate was analyzed with gamma spectrometry. Auto radiographic images were obtained from coronal slices of frozen heads of mice administered with (201)Tl or (54)Mn. The transection of the olfactory nerve fibers was confirmed with a neuronal tracer. The transport of intranasal administered (201)Tl/(54)Mn to the olfactory bulb was significantly reduced by the transection of olfactory nerve fibers. The olfactory nerve transection also significantly inhibited the accumulation of fluoro-ruby in the olfactory bulb. Findings indicate that thallium is transported by the olfactory nerve fibers to the olfactory bulb in mice. The assessment of thallium transport following head injury may provide a new diagnostic method for the evaluation of olfactory nerve injury.     

Odor detection ability and thallium-201 transport in the olfactory nerve of traumatic olfactory-impaired mice

Although olfactory nerve damage is a contributing factor in the diagnosis of posttraumatic olfactory loss, at present, there are no methods to directly assess injury to these nerves. We have shown that following olfactory nerve injury in mice, thallium-201 (201 Tl) transport from the nasal cavity to the olfactory bulb decreases. To determine if olfactory function after nerve injury could be assessed with nasal administration of 201 Tl, we measured the correlation between odor detection ability (ODA) and the rate of transport of 201 Tl in olfactory nerves. Both ODA and 201 Tl transport were measured after bilateral olfactory nerve transection for a 4-week period. Cycloheximide solution was used for ODA against tap water. 201 Tl transport was measured as the ratio of radioactivity in the nasal cavity and olfactory bulb with gamma spectrometry. There was a significant correlation between ODA and the rate of 201 Tl transport in the olfactory nerve. These findings suggest that olfactory function after nerve injury can be objectively evaluated with the nasal administration of 201 Tl.     

IL-2 withdrawal induces HTLV-1 expression through p38 activation in ATL cell lines

Expression of human T-cell leukemia virus type-1 (HTLV-1) in adult T-cell leukemia (ATL) cells is known to be marginal in vivo and inducible in short-term culture. In this study, we demonstrated that withdrawal of interleukin (IL)-2 from IL-2-dependent ATL cell lines resulted in induction of HTLV-1 mRNA and protein expression, and that viral induction was associated with phosphorylation of the stress kinase p38 and its downstream CREB. Pharmacological inhibitors of the p38 pathway suppressed viral expression induced by IL-2 depletion. These results indicate that the stress-induced p38 pathway might up-regulate HTLV-1 gene expression through at least CREB activation.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.     

Assessment of Olfactory Nerve by SPECT-MRI Image with Nasal Thallium-201 Administration in Patients with Olfactory Impairments in Comparison to Healthy Volunteers

Purpose

The aim of this study was to assess whether migration of thallium-201 (²⁰¹Tl) to the olfactory bulb were reduced in patients with olfactory impairments in comparison to healthy volunteers after nasal administration of ²⁰¹Tl.

Procedures

10 healthy volunteers and 21 patients enrolled in the study (19 males and 12 females; 26–71 years old). The causes of olfactory dysfunction in the patients were head trauma (n = 7), upper respiratory tract infection (n = 7), and chronic rhinosinusitis (n = 7). ²⁰¹TlCl was administered unilaterally to the olfactory cleft, and SPECT-CT was conducted 24 h later. Separate MRI images were merged with the SPECT images. ²⁰¹Tl olfactory migration was also correlated with the volume of the olfactory bulb determined from MRI images, as well as with odor recognition thresholds measured by using T&T olfactometry.

Results

Nasal ²⁰¹Tl migration to the olfactory bulb was significantly lower in the olfactory-impaired patients than in healthy volunteers. The migration of ²⁰¹Tl to the olfactory bulb was significantly correlated with odor recognition thresholds obtained with T&T olfactometry and correlated with the volume of the olfactory bulb determined from MRI images when all subjects were included.

Conclusions

Assessment of the ²⁰¹Tl migration to the olfactory bulb was the new method for the evaluation of the olfactory nerve connectivity in patients with impaired olfaction.     

Inhibition of G protein-activated inwardly rectifying K+ channels by different classes of antidepressants

Various antidepressants are commonly used for the treatment of depression and several other neuropsychiatric disorders. In addition to their primary effects on serotonergic or noradrenergic neurotransmitter systems, antidepressants have been shown to interact with several receptors and ion channels. However, the molecular mechanisms that underlie the effects of antidepressants have not yet been sufficiently clarified. G protein-activated inwardly rectifying K(+) (GIRK, Kir3) channels play an important role in regulating neuronal excitability and heart rate, and GIRK channel modulation has been suggested to have therapeutic potential for several neuropsychiatric disorders and cardiac arrhythmias. In the present study, we investigated the effects of various classes of antidepressants on GIRK channels using the Xenopus oocyte expression assay. In oocytes injected with mRNA for GIRK1/GIRK2 or GIRK1/GIRK4 subunits, extracellular application of sertraline, duloxetine, and amoxapine effectively reduced GIRK currents, whereas nefazodone, venlafaxine, mianserin, and mirtazapine weakly inhibited GIRK currents even at toxic levels. The inhibitory effects were concentration-dependent, with various degrees of potency and effectiveness. Furthermore, the effects of sertraline were voltage-independent and time-independent during each voltage pulse, whereas the effects of duloxetine were voltage-dependent with weaker inhibition with negative membrane potentials and time-dependent with a gradual decrease in each voltage pulse. However, Kir2.1 channels were insensitive to all of the drugs. Moreover, the GIRK currents induced by ethanol were inhibited by sertraline but not by intracellularly applied sertraline. The present results suggest that GIRK channel inhibition may reveal a novel characteristic of the commonly used antidepressants, particularly sertraline, and contributes to some of the therapeutic effects and adverse effects.     

Estimation of hydroxyl radical generation by salicylate hydroxylation method in multiple organs of mice exposed to whole-body X-ray irradiation

Appropriate experimental conditions for the estimation of hydroxyl radical generation by salicylate hydroxylation were determined for multiple organs of X-irradiated mice in vivo. The in vitro experiments showed that there were significant correlations between the salicylic acid (SA) concentration, the amount of 2,3-dihydroxy benzoic acid (2,3-DHBA) and the X-ray exposure dose, and we obtained two linear-regression equations to calculate the amounts of hydroxyl radicals generated by the X-irradiation. The optimum dosage of SA and the appropriate sampling time for in vivo experiments was determined, and significant increases in the ratio of 2,3-DHBA to SA were detected in several organs of mice after X-irradiation. The hydroxyl radical equivalents of the 2,3-DHBA increases were also calculated. Our results clearly demonstrated the usefulness of the salicylate hydroxylation method in estimating hydroxyl radical generation in multiple organs in vivo.