Development of an alpha-fetoprotein and Golgi protein 73 multiplex detection assay using xMAP technology.

AIM OF THE STUDY: Development of a new method to simultaneously detect Alpha-fetoprotein (AFP) and Golgi protein 73 (GP73) from peripheral blood. MATERIAL AND METHODS: Anti human AFP and GP73 monoclonal antibodies was used to develop a sandwich immunity reaction using xMAP technology for the simultaneous detection of plasma AFP and GP73. The assay evaluated the sensitivity, cross reactivity, range of detection, precision, recovery and linearity dilution effect. The assay utilized plasma samples and compared its performance with commercially available Enzyme Linked Immunosorbent Assay (ELISA) kits. RESULTS: The assay was successful in detecting AFP and GP73 simultaneously. Validation experiments demonstrated the limit of detection was AFP 0.006 µg/l and GP73 0.482 µg/l. The functional sensitivity was AFP 0.010 µg/l and GP73 0.640 µg/l. The range of detection was AFP 0.01-50 µg/l and GP73 0.64-100 µg/l. No cross reactivity was observed. The intra- and inter-assay variation for AFP was 0.19-3.46% and 3.1-5.8% and for GP73 was 1.5-3.2% and 1.1-7.6% respectively. The recovery for AFP was 96-106% and GP73 was 89-110%. 80 clinical plasma samples from healthy controls, and patients with liver cirrhosis and Hepatocellular Carcinoma (HCC) were evaluated. For healthy controls (n = 25), the AFP was 2.40 (1.55, 3.30) µg/l and GP73 was 42.60 (39.10, 57.40) µg/l. For patients with liver cirrhosis (n = 19), the AFP was 2.60 (1.70, 4.20) µg/l and GP73 was 136.10 (92.10, 261.70) µg/l, and for HCC patients (n = 36), the AFP was 13.65 (3.35, 158.88) µg/l and GP73 was 186.25 (96.73, 262.03) µg/l. The new assay demonstrated a good correlation with commercially available ELISA kits (correlation coefficients (r) were 0.997 (AFP, p < 0.001) and 0.959 (GP73, p < 0.001). CONCLUSIONS: The method demonstrated a sensitive, effective and accurate method for the simultaneous detection of AFP and GP73, and could be used clinically for routine detection and monitoring of patients with chronic hepatitis B.

Traumatic rupture of the neurocentral synchondrosis of the axis in a child.

The authors report the first case of unilateral traumatic rupture of the C-2 neurocentral synchondrosis. A 26-month-old child was in a vehicular collision that caused his head to be rotated sharply to the left with the neck flexed. He had severe neck pain but was neurologically normal. Computerized tomography scanning showed rupture of the left C-2 neurocentral synchondrosis, a right C-2 pars interarticularis fracture, and anterior angulation of C-2 on C-3. The neck injury was unrecognized until postinjury Day 9 when an MRI study showed a tear of the posterior longitudinal ligament at C2-3 and separation of the C-2 body from the inferior anular epiphysis. A second CT showed widening of the synchondrosis fracture, increased angulation of C-2 on C-3, and distraction of the right C-2 pars fracture. The mechanism of the neurocentral synchondrosis fracture is thought to be hyperflexion-axial loading combined with leftward rotation, which provided the lateral force that overcame the cartilaginous synchondrosis and extruded the lateral mass. The patient underwent open reduction and posterior fusion of C1-3, and was maintained in a halo jacket for 4 months, when CT scans demonstrated solid C1-C3 fusion and ossification of the injured synchondrosis. Unilateral traumatic rupture of the C-2 neurocentral synchondrosis is one component of several injuries involving C-2 sustained before synchondrosis closure. The resulting C2-3 relationship is highly unstable. Reduction and C1-C3 fusion are necessary in patients with significant displacement of the adjacent bony units.

Dimethyl sulfoxide provides neuroprotection in a traumatic brain injury model.

PURPOSE: The objective of this study was to evaluate the neuroprotective potential of the antioxidant, curcumin compared to alpha-tocopherol in a rat model of traumatic brain injury (TBI). METHODS: Male Sprague-Dawley rats were administered curcumin (3, 30, 300 mg/kg), alpha-tocopherol (100 mg/kg), DMSO vehicle, or saline, 30 min prior to and 30 and 90 min after moderate lateral fluid percussion TBI. Rats were euthanized at 24 hours after injury and coronal brain sections were stained with Fluoro-Jade to identify degenerating neurons. Degenerating neurons in the CA2-3 sector of the dorsal hippocampus were quantified in 10 sections spaced 300 microm apart in each rat. RESULTS: One way ANOVA revealed a significant difference (p = 0.01) between groups. The curcumin, alpha-tocopherol, and DMSO groups had significantly reduced numbers of degenerating neurons compared to the saline-treated group. No significant differences were observed between any of the drug treatment groups or the DMSO group. CONCLUSIONS: Since protection in the DMSO vehicle group was equal to that of the experimental groups, no conclusions about neuroprotection regarding alpha-tocopherol or curcumin can be made from this study. The results suggest that DMSO may be acting as an overriding neuroprotectant in this experiment. We conclude that DMSO is a viable neuroprotective agent against secondary cell death in TBI.

Assessment of proton microbeam analysis of 11B for quantitative microdistribution analysis of boronated neutron capture agents in biological tissues.

The (11)B(p,alpha)(8)Be* nuclear reaction was assessed for its ability to quantitatively map the in vivo subcellular distribution of boron within gliosarcomas treated with a boronated neutron capture therapy agent. Intracranial 9L gliosarcomas were produced in Fischer 344 rats. Fourteen days later, the majority of the rats were treated with f-boronophenylalanine and killed humanely 30 or 180 min after intravenous injection. Freeze-dried tumor cryosections were imaged using the (11)B(p,alpha)(8)Be* nuclear reaction and proton microbeams obtained from the nuclear microprobe at Lawrence Livermore National Laboratory. The (11)B distributions within cells could be imaged quantitatively with spatial resolutions down to 1.5 microm, minimum detection limits of 0.8 mg/kg, and acquisition times of several hours. These capabilities offer advantages over alpha-particle track autoradiography, electron energy loss spectroscopy, and secondary ion mass spectrometry (SIMS) for quantification of (11)B in tissues. However, the spatial resolution, multi-isotope capability, and analysis times achieved with SIMS are superior to those achieved with (11)B(p,alpha)(8)Be* analysis. When accuracy in quantification is crucial, the (11)B(p,alpha)(8)Be* reaction is well suited for assessing the microdistribution of (11)B. Otherwise, SIMS may well be better suited to image the microdistribution of boron associated with neutron capture therapy agents in biological tissues.

Syntheses and preliminary biological studies of four meso-Tetra[(nido-carboranylmethyl)phenyl]porphyrins.

Two meso-tetra[(nido-carboranylmethyl)phenyl]porphyrins (para- and meta-regioisomers) and their corresponding Zn(II) complexes have been synthesized with the aim of studying the effect of carborane distribution and metalation on the biological properties of this series of compounds. In vitro cell toxicity, uptake/efflux, and subcellular localization using rat 9L, mouse B16 and/or human U-373MG cells were evaluated. All four amphiphilic porphyrins display very low cytotoxicities and time- and concentration-dependent uptake by cells, which is influenced by serum proteins. Preliminary subcellular localization studies suggest that one of these compounds localizes in close proximity to the cell nucleus. All four nido-carboranylporphyrins show promise as boron-carriers for the boron neutron capture therapy of cancers, particularly the metal-free nido-carboranylporphyrins 5 and 12, which are able to deliver higher amount of boron to cells in vitro than the corresponding zinc complexes.