Carbamylated erythropoietin ameliorates cyclosporine nephropathy without stimulating erythropoiesis

The introduction of cyclosporine (CsA) has improved graft survival, but it causes nephropathy, which limits its clinical utility. Recently, we reported that carbamylated erythropoietin (CEPO) protected kidneys from ischemia reperfusion injury as well as EPO. To investigate the clinical applications of CEPO, we next evaluated the long-term therapeutic effect of CEPO using a CsA-induced nephropathy model. CsA caused renal dysfunction, while EPO/CEPO administration significantly improved renal function. EPO treatment significantly increased Hb concentration, while CEPO treatment neither enhanced nor reduced Hb concentration. CsA treatment induced tubular apoptosis, while EPO/CEPO administration inhibited it and increased PI3 kinase activation and Akt phosphorylation. In parallel, morphological assessment revealed that EPO/CEPO significantly reduced CsA-induced interstitial fibrosis and inhibited interstitial macrophage infiltration. In addition, real-time RT-PCR demonstrated that cortical mRNA levels of TGF-β1 and type I collagen were suppressed in the EPO/CEPO group. These results suggest a new therapeutic approach using CEPO to protect kidneys from CsA-induced nephropathy.     

Actions of Midazolam on Excitatory Transmission in Dorsal Horn Neurons of Adult Rat Spinal Cord

Background: Although intrathecal administration of midazolam, a water-soluble imidazobenzodiazepine derivative, has been found to produce analgesia, how it exerts this effect at the neuronal level in the spinal cord is not fully understood.

Methods: The effects of midazolam on electrically evoked and spontaneous excitatory transmission were examined in lamina II neurons of adult rat spinal cord slices using the whole cell patch clamp technique.

Results: Bath-applied midazolam (1 [mu]m) diminished A[delta]- and C-fiber evoked polysynaptic excitatory postsynaptic currents in both amplitude and integrated area. However, it affected neither A[delta]- and C-fiber evoked monosynaptic excitatory postsynaptic currents in amplitude nor miniature excitatory postsynaptic currents in amplitude, frequency, and decay time constant. In the presence of a benzodiazepine receptor antagonist, flumazenil (5 [mu]m), midazolam (1 [mu]m) did not diminish A[delta]-fiber evoked polysynaptic excitatory postsynaptic currents, suggesting that midazolam modulate the [gamma]-aminobutyric acid interneurons in the dorsal horn.     

Pulmonary endothelial chimerism after hematopoietic stem cell transplantation

Purpose

Few studies have investigated pulmonary endothelial chimerism after hematopoietic stem cell transplantation. In the present study, we investigated pulmonary endothelial chimerism using the ABH histo-blood group antigen as an identifying marker in cases of ABO-incompatible hematopoietic stem cell transplantation.

Methods

Sixteen lung samples were analyzed. Of these, seven were explanted lungs from lung transplant recipients with severe pulmonary chronic graft-versus-host disease (GVHD). The remaining nine were autopsy samples from patients who died from various causes, and six of these nine cases had a diagnosis of pulmonary chronic GVHD. The ABH histo-blood group antigen was used to differentiate donor cells from recipient cells immunohistochemically. We estimated the percentage of vessels positive for donor blood group antigens in comparison with the total number of vessels.

Results

Donor blood group antigens were expressed in the endothelium of 13 samples, all of which were pathologically diagnosed with pulmonary chronic GVHD. The proportion of vessels with donor group antigens ranged from 0.1 to 17.5%. In contrast, no chimeric vessels were observed in the three samples without pulmonary chronic GVHD.

Conclusions

Our results demonstrate that circulating stem cells engraft into the endothelium to a considerable extent in pulmonary chronic GVHD.

     

Possible involvement of cationic‐drug sensitive transport systems in the blood‐to‐brain influx and brain‐to‐blood efflux of amantadine across the blood–brain barrier

The purpose of this study was to characterize the brain‐to‐blood efflux transport of amantadine across the blood–brain barrier (BBB). The apparent in vivo efflux rate constant for [³H]amantadine from the rat brain (k_eff) was found to be 1.53 × 10^‐2 min^‐1 after intracerebral microinjection using the brain efflux index method. The efflux of [³H]amantadine was inhibited by 1‐methyl‐4‐phenylpyridinium (MPP⁺), a cationic neurotoxin, suggesting that amantadine transport from the brain to the blood across the BBB potentially involves the rat plasma membrane monoamine transporter (rPMAT). On the other hand, other selected substrates for organic cation transporters (OCTs) and organic anion transporters (OATs), as well as inhibitors of P‐glycoprotein (P‐gp), did not affect the efflux transport of [³H]amantadine. In addition, in vitro studies using an immortalized rat brain endothelial cell line (GPNT) showed that the uptake and retention of [³H]amantadine by the cells was not changed by the addition of cyclosporin, which is an inhibitor of P‐gp. However, cyclosporin affected the uptake and retention of rhodamine123. Finally, the initial brain uptake of [³H]amantadine was determined using an in situ mouse brain perfusion technique. Notably, the brain uptake clearance for [³H]amantadine was significantly decreased with the co‐perfusion of quinidine or verapamil, which are cationic P‐gp inhibitors, while MPP⁺ did not have a significant effect. It is thus concluded that while P‐gp is not involved, it is possible that rPMAT and the cationic drug‐sensitive transport system participate in the brain‐to‐blood efflux and the blood‐to‐brain influx of amantadine across the BBB, respectively. Copyright © 2014 John Wiley & Sons, Ltd.     

Imaging endogenous gene expression in brain cancer in vivo with 111In-peptide nucleic acid antisense radiopharmaceuticals and brain drug-targeting technology.

Imaging endogenous gene expression with sequence-specific antisense radiopharmaceuticals is possible if the antisense agent is enabled to traverse the biologic membrane barriers that separate the blood compartment from messenger RNA (mRNA) molecules in the cytoplasm of the target cell. The present studies were designed to image endogenous gene expression in brain cancer using peptide nucleic acid (PNA) antisense agents that were modified to allow for (a) chelation of the 111In radionuclide and (b) attachment to a brain targeting system, which delivers the PNA across both the blood-brain barrier (BBB) and the tumor cell membrane. METHODS: PNAs were designed that were antisense to either the rat glial fibrillary acidic protein (GFAP) mRNA or the rat caveolin-1alpha (CAV) mRNA. The PNA contained an amino-terminal diethylenetriaminepentaacetic acid moiety to chelate 111In and a carboxyl-terminal epsilon-biotinyl lysine residue, which enabled attachment to the delivery system. The latter comprised streptavidin (SA) and the murine OX26 monoclonal antibody to the rat transferrin receptor (TfR), which were joined by a thiol-ether linker. Control PNAs were not conjugated to SA-OX26. Brain tumors developed after the intracerebral injection of rat RG2 glial cells in adult Fischer CD344 rats. GFAP and CAV gene expression in the tumor in vivo was monitored by confocal microscopy and Northern blotting with GFAP and CAV complementary DNAs. RESULTS: If the PNA was not targeted to the TfR, then no imaging of any brain structures was possible, owing to the absence of PNA transport across the BBB. Conjugation of the 111In-GFAP-PNA to the SA-OX26 delivery system did not image brain cancer, owing to the downregulation of the GFAP mRNA in brain glial tumors. In contrast, brain cancer was selectively imaged with the 111In-CAV-PNA conjugated to SA-OX26 owing to upregulation of CAV gene expression in brain cancer. CONCLUSION: Imaging endogenous gene expression in vivo with PNA antisense radiopharmaceuticals is possible if drug-targeting technology is used. Attachment of the PNA antisense agent to the targeting ligand enables the antisense radiopharmaceutical to traverse biologic membrane barriers and access intracellular target mRNA molecules.     

Comparative Study of Cisplatin and Carboplatin on Pharmacokinetics,Nephrotoxicity and Effect on Renal Nuclear DNA Synthesis in Rats

Abstract: To clarify the difference in nephrotoxicity between cisplatin and carboplatin, the pharmacokinetics of platinum, renal function and nuclear DNA synthesis in renal cortical and outer medullary cells were studied in rats which had received cisplatin or carboplatin. Male Sprague-Dawley rats were given either cisplatin or carboplatin intravenously at an equi-toxic dose (LD₁₀ or LD₅₀) and were killed at various times within 7 days after the injection. Cisplatin bound to plasma proteins more avidly than carboplatin. Much more platinum was detectable in the renal nuclei after cisplatin injection than after carboplatin injection. BUN and serum creatinine levels in the rats treated with 8.5 mg/kg of cisplatin were significantly higher than in those treated with 100 mg/kg of carboplatin. Cisplatin markedly suppressed the renal nuclear DNA synthesis both in vivo and in vitro, when compared with carboplatin. It is concluded that the differences in nephrotoxicity between cisplatin and carboplatin are related to their different inhibitory effects on nuclear DNA synthesis in the renal cells.     

Proteomics-based approach identifying autoantibody against peroxiredoxin VI as a novel serum marker in esophageal squamous cell carcinoma.

PURPOSE: Detection of novel tumor-related antigens and autoantibodies will aid in diagnosis of early-stage cancer and in development of more effective immunotherapies. The purpose of this study was to identify novel tumor antigens in an esophageal squamous cell carcinoma (ESCC) cell line (TE-2) and related autoantibodies in sera from patients with ESCC using a proteomics-based approach. EXPERIMENTAL DESIGN: TE-2 proteins were separated by two-dimensional PAGE, followed by Western blot analysis in which sera of patients with ESCC, healthy controls, and patients with other cancers were tested for primary antibodies. Positive spots were excised from silver-stained gels and analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS). RESULTS: Sera from patients with ESCC yielded multiple spots, one of which was identified as peroxiredoxin (Prx) VI by MALDI-TOF/TOF MS. Western blot analysis against recombinant Prx VI showed reactivity in sera from 15 of 30 (50%) patients with ESCC and 2 of 30 (6.6%) healthy individuals. Autoantibody against Prx VI was found in sera from 1 of 30 (3.3%) patients with other types of cancer (colon cancer). CONCLUSION: We have identified for the first time an autoantibody against Prx VI in ESCC patients. The proteomic approach implemented here offers a powerful tool for identifying novel serum markers that may display clinical usefulness against cancer.     

Utility of the Japanese GFR estimation equation for evaluating potential donor kidney function

Background

In Japan, the number of living kidney transplantations has increased each year, and an accurate evaluation of renal function must be conducted before donation to minimize the risk to donors. Recently, the Japanese Society of Nephrology issued a new equation for estimating glomerular filtration rate (eGFR) in Japanese people. This study compared the accuracy of eGFR and creatinine clearance (Ccr) values with that of inulin clearance (Cin) for assessing renal function in kidney donors.

Methods

Clinical data were analyzed for 85 potential living kidney donors who had undergone routine measured GFR (mGFR) and Ccr measurements from October 2006 to November 2008 at a single center. Inulin clearance, representing the mGFR, was determined by standard method. The eGFR was calculated as: eGFR = 194 × Scr^?1.094 × Age^?0.287 (for females, ×0.739).

Results

Mean mGFR was 96.1 ± 14.7 (range 67.8–126.8); mean eGFR, 72.6 ± 12.7 (range 50.1–107.1); and mean Ccr, 117.3 ± 22.4 (range 35.1–170.1), in units of ml/min/1.73 m² for each. Relative to mGFR, the correlation coefficient for Ccr was 0.496, and the mean difference between the two values was 21.1 ml/min/1.73 m² (23.2%), with a root-mean square error (RMSE) of 19.6. The correlation coefficient between eGFR and mGFR was 0.502, and the mean difference between the two values was ?23.5 (23.7%), with a RMSE of 11.0. Bland–Altman plots showed that Ccr overestimated mGFR in 90.6% of cases, whereas eGFR underestimated mGFR in 95.3% of cases.

Conclusion

Ccr and eGFR values did not accurately estimate mGFR in Japanese living kidney donors.