(R)-[11C]verapamil is selectively transported by murine and human P-glycoprotein at the blood–brain barrier,and not by MRP1 and BCRP

IntroductionPositron emission tomography (PET) with [¹¹C]verapamil, either in racemic form or in form of the (R)-enantiomer, has been used to measure the functional activity of the adenosine triphosphate-binding cassette (ABC) transporter P-glycoprotein (Pgp) at the blood–brain barrier (BBB). There is some evidence in literature that verapamil inhibits two other ABC transporters expressed at the BBB, i.e. multidrug resistance protein 1 (MRP1) and breast cancer resistance protein (BCRP). However, previous data were obtained with micromolar concentrations of verapamil and do not necessarily reflect the transporter selectivity of verapamil at nanomolar concentrations, which are relevant for PET experiments. The aim of this study was to assess the selectivity of verapamil, in nanomolar concentrations, for Pgp over MRP1 and BCRP.MethodsConcentration equilibrium transport assays were performed with [³H]verapamil (5 nM) in cell lines expressing murine or human Pgp, human MRP1, and murine Bcrp1 or human BCRP. Paired PET scans were performed with (R)-[¹¹C]verapamil in female FVB/N (wild-type), Mrp1^(?/?), Mdr1a/b^(?/?), Bcrp1^(?/?) and Mdr1a/b^(?/?)Bcrp1^(?/?) mice, before and after Pgp inhibition with 15 mg/kg tariquidar.ResultsIn vitro transport experiments exclusively showed directed transport of [³H]verapamil in Mdr1a- and MDR1-overexpressing cells which could be inhibited by tariquidar (0.5 μM). In PET scans acquired before tariquidar administration, brain-to-blood ratio (K_b,brain) of (R)-[¹¹C]verapamil was low in wild-type (1.3 ± 0.1), Mrp1^(?/?) (1.4 ± 0.1) and Bcrp1^(?/?) mice (1.8 ± 0.1) and high in Mdr1a/b^(?/?) (6.9 ± 0.8) and Mdr1a/b^(?/?)Bcrp1^(?/?) mice (7.9 ± 0.5). In PET scans after tariquidar administration, K_b,brain was significantly increased in Pgp-expressing mice (wild-type: 5.0 ± 0.3-fold, Mrp1^(?/?): 3.2 ± 0.6-fold, Bcrp1^(?/?): 4.3 ± 0.1-fold) but not in Pgp knockout mice (Mdr1a/b^(?/?) and Mdr1a/b^(?/?)Bcrp1^(?/?)).ConclusionOur combined in vitro and in vivo data demonstrate that verapamil, in nanomolar concentrations, is selectively transported by Pgp and not by MRP1 and BCRP at the BBB, which supports the use of (R)-[¹¹C]verapamil or racemic [¹¹C]verapamil as PET tracers of cerebral Pgp function.     

Saturation‐recovery metabolic‐exchange rate imaging with hyperpolarized [1‐13C] pyruvate using spectral‐spatial excitation

Within the last decade hyperpolarized [1‐¹³C] pyruvate chemical‐shift imaging has demonstrated impressive potential for metabolic MR imaging for a wide range of applications in oncology, cardiology, and neurology. In this work, a highly efficient pulse sequence is described for time‐resolved, multislice chemical shift imaging of the injected substrate and obtained downstream metabolites. Using spectral‐spatial excitation in combination with single‐shot spiral data acquisition, the overall encoding is evenly distributed between excitation and signal reception, allowing the encoding of one full two‐dimensional metabolite image per excitation. The signal‐to‐noise ratio can be flexibly adjusted and optimized using lower flip angles for the pyruvate substrate and larger ones for the downstream metabolites. Selectively adjusting the excitation of the down‐stream metabolites to 90° leads to a so‐called “saturation‐recovery” scheme with the detected signal content being determined by forward conversion of the available pyruvate. In case of repetitive excitations, the polarization is preserved using smaller flip angles for pyruvate. Metabolic exchange rates are determined spatially resolved from the metabolite images using a simplified two‐site exchange model. This novel contrast is an important step toward more quantitative metabolic imaging. Goal of this work was to derive, analyze, and implement this “saturation‐recovery metabolic exchange rate imaging” and demonstrate its capabilities in four rats bearing subcutaneous tumors. Magn Reson Med, 2013. © 2012 Wiley Periodicals, Inc.     

The continuous epidural infusion of ropivacaine 0.1% with 0.5 μg·mL−1 sufentanil provides effective postoperative analgesia after total hip replacement: a pilot study

PURPOSE: To assess the analgesic efficacy and functional outcome of postoperative epidural infusion of ropivacaine combined with sufentanil in a randomized, controlled trial. METHODS: Thirty-two ASA I-III patients undergoing elective total hip replacement (THR) were included. Lumbar epidural block using 0.75% ropivacaine was combined with either propofol sedation or general anesthesia for surgery. On arrival in the recovery room, the epidural infusion was commenced at a rate in mL calculated as follows: (height in cm - 100) x 0.1. Eleven patients received an epidural infusion of ropivacaine 0.1% with 0.5 microg x mL(-1) sufentanil (Group R+S0.5), ten patients ropivacaine 0.1% with 0.75 microg x mL(-1) sufentanil (Group R+S0.75), and 11 patients ropivacaine 0.1% with 1 microg x mL(-1) sufentanil (Group R+S1) over a postoperative study period of 44 hr. All patients had access to iv piritramide via a patient-controlled analgesia (PCA) device. Postel-Merle-d'Aubigné scoring system (PMA score) was assessed preoperatively, three weeks after surgery, and three months after surgery by an orthopedic surgeon blinded to study group. RESULTS: Motor block was negligible in all three groups. After eight hours of epidural infusion, sensory block had regressed completely in all patients. There was no significant difference with regard to visual analogue scale (VAS) scores (at rest: P = 0.55, on movement: P = 0.63), consumption of rescue medication (P = 0.99), patient satisfaction (P = 0.22), and the incidence of adverse events. All treatment regimens provided effective postoperative analgesia with only a minimal use of supplemental opioid PCA. There was no difference between groups regarding orthopedic PMA score (pain: P = 0.24, mobility: P = 0.65, and ability to walk: P = 0.44). CONCLUSION: Ropivacaine 0.1% with 0.5 microg x mL(-1) sufentanil for postoperative analgesia after THR provides efficient pain relief and, compared with 0.75 and 1 microg x mL(-1) sufentanil, reduces sufentanil consumption without compromise in patient satisfaction, VAS scores, and functional outcome.     

Time course of reversed cardiac remodeling after pulmonary endarterectomy in patients with chronic pulmonary thromboembolism

To evaluate the time course of reversed remodeling after pulmonary endarterectomy (PEA) in patients with chronic thromboembolic pulmonary hypertension(CTPEH), we studied 22 patients (age: 60 ± 13 years) with MRI immediately before, 1 month, 3 months, and 6 months after PEA. MRI included assessment of biventricular function, aortic and pulmonary artery(PA) flow, and right ventricular (RV) overload using the ratio of RV-to-biventricular diameter. Except in one patient, who died 2 months post-surgery, clinical improvement occurred early after PEA (NYHA class: 3.3 ± 0.6 to 1.5 ± 0.8, p < 0.0001) with a decrease of systolic pulmonary artery pressures (79 ± 14 to 44 ± 14 mmHg, p < 0.0001). At 1 month post PEA, RV end-diastolic volumes decreased (198 ± 72 to 137 ± 59 ml, p < 0.0001), and the RV ejection fraction (EF) improved (31 ± 9 to 47 ± 10%, p < 0.0001). No further significant improvement in pulmonary pressures or RV function occurred at 3 months or 6 months. Although no significant change was found in LV volumes or function, aortic flow increased early after surgery. PEA had only a beneficial effect on right PA flow. RV overload decreased early after PEA (ratio RV-to-biventricular diameter: before: 0.67 ± 0.04, after: 0.54 ± 0.06, p < 0.0001), showing a good correlation with the improvement in RVEF (r = 0.7, P < 0.0001). In conclusion, reversed cardiac remodeling occurs early after PEA, to slow down after 1 month. At 6 months, cardiac remodeling is incomplete as witnessed by low-normal RV function and residually elevated PA pressures.     

Mycophenolate mofetil significantly reduces leukocyte graft infiltration after heterotopic cardiac transplantation in a rat model: comparative study with cyclosporine and FK 506.

BACKGROUND: The purpose of the study was to evaluate the effects of cyclosporine (CsA), FK 506 and mycophenolate mofetil (MMF) on graft-infiltrating leukocytes (CD4, CD8, CD11a, CD18) after cardiac transplantation in rats. METHODS: Three hundred forty animals were transplanted and randomly divided into 4 groups: CsA, 3 mg/kg/d (n = 74); MMF, 40 mg/kg/d (n = 96); FK 506, 0.3 mg/kg/d (n = 96); and a control group receiving no immunosuppressive therapy (n = 74). Three or 4 animals from each group were killed at intervals of 1 to 4 days up to Day 60. Immunohistochemistry was performed using monoclonal antibodies (MAb) against CD4, CD8, CD11a and CD18. Positively stained cells were analyzed in the perivascular space (PVS) of intra- and epicardial arteries. Statistical analysis was performed using area-under-the-curve assessment with an extended t-test. RESULTS: CsA and FK 506 reduced the presence graft-infiltrating leukocytes (CD4, CD8, CD11a, CD18) in the PVS of intra- and epicardial arteries when compared with control animals. MMF therapy resulted in a further significant reduction in infiltrating leukocytes when compared with the 2 calcineurin inhibitors. MMF had a faster onset of action than the calcineurin inhibitors. CsA and FK 506 required 12 to 20 additional days to achieve the reducing effect of graft infiltration seen in MMF-treated animals. CONCLUSION: MMF possesses potent infiltration-blocking properties and its application leads to a greater reduction of cellular infiltration in the course of transplant rejection when compared with calcineurin inhibitors.     

Matched analysis of nonoperative management vs immediate appendectomy for perforated appendicitis

Background

The role of nonoperative therapy vs immediate appendectomy in the management of children with perforated appendicitis remains undefined. The objective of this study was to rigorously compare these management options in groups of patients with matched clinical characteristics.

Methods

Multicenter case-control study was conducted from 1998 to 2003. We compared patients treated nonoperatively vs those undergoing appendectomy to identify differences in 12 clinical parameters. We then generated a second control group of patients matched for these variables and compared the following outcomes in these clinically similar groups: complication rate, abscess rate, and length of stay (LOS). Analysis was performed according to intention-to-treat principles, using χ², Fisher exact, and Student t tests.

Results

The only significant difference between patients treated nonoperatively and those treated by appendectomy was the duration of pain on presentation (6.8 vs 3.1 days of pain).We created a second control group of patients undergoing immediate appendectomy matched on duration of pain on presentation to patients treated nonoperatively. These groups continued to be clinically comparable for the other 11 parameters. Compared to this matched control group, the nonoperative group had fewer complications (19% vs 43%, P < .01), fewer abscesses (4% vs 24%, P < .01), and a trend for shorter LOS (6.5 ± 5.7 vs 8.8 ± 6.7 days, P = .08).

Conclusions

When nonoperative management for perforated appendicitis was studied using appropriately matched clinical controls, we found that it resulted in a lower complication rate and shorter LOS in the subset of patients presenting with a long duration of pain. Our data suggest that nonoperative management should be prospectively evaluated in children with perforated appendicitis presenting with a history of pain exceeding 5 days.     

Long-term survival after radical resection for pancreatic head and ampullary cancer: a potential role for the EGF-R

BACKGROUND/AIM: Pancreatic cancer has a dismal prognosis. Ampullary cancer (defined as cancer of the ampulla of Vater or the distal common bile duct) has a better prognosis and is thought to be a biologically different tumor. The aim of this study was to find factors that could predict survival after radical (R-0) resection for pancreatic head and ampullary cancers. METHODS: We analyzed clinical and pathological data from 93 patients who underwent a true R-0 resection for pancreatic head or ampullary cancer. Furthermore, we performed a tissue microarray protein expression analysis for several growth factor receptors and oncogenes: HER-2, EGF-R, ER, PR, C-myc, p53, p16, RB-1, and chromogranin A as a neuroendocrine differentiation marker. RESULTS: Median survival (14 vs. 42 months) and time to recurrence (16 vs. 42 months) were significantly longer for ampullary than for pancreatic head cancers. Preoperative pain, perineural invasion, lymph node metastasis, and tumor differentiation grade are indicators of a poor survival. No differences in protein expression were found between groups, except for EGF-R which was expressed more in pancreatic head cancers (p = 0.026). CONCLUSIONS: Outcomes for ampullary cancers are better than for pancreatic head cancers. This different biological behavior can possibly be explained by differences in EGF-R expression.     

Quantitative analysis of EBV-specific CD4/CD8 T cell numbers, absolute CD4/CD8 T cell numbers and EBV load in solid organ transplant recipients with PLTD

Post transplant lymphoproliferative disease (PTLD) in solid organ transplant (SOT) recipients is assumed to be the result of impaired Epstein-Barr Virus (EBV)-specific cellular immunity. We analyzed the absolute CD4 and CD8 T cell counts as well as the EBV-specific CD4 and CD8 T cell responses in relation to EBV load in SOT recipients with PTLD. A prospective, single center study was initiated and 10 immunosuppressed patients with diagnosis of PTLD were analyzed and compared to 3 patients without PTLD (2 SOT recipients with EBV-reactivation, 1 patient with Infectious Mononucleosis) and 6 healthy EBV positive controls. EBV-specific CD8 T cells were enumerated using HLA class I tetramers and the IFN-gamma cytokine secretion assay. EBNA1-specific CD4 T cells were analyzed after protein stimulation and EBV load was quantified by real-time PCR. Absolute CD8 T cell counts were highly variable in all 19 cases analyzed. In contrast, the absolute EBV-specific CD8 T cell count was found to be low in 7/9 patients with PTLD (<5/microl whole blood). These frequencies were similar to absolute EBV-specific CD8 T cell numbers observed in healthy EBV positive donors, but much lower compared to patients with EBV reactivation but no PTLD. Absolute CD4 T cell counts were significantly lower in PTLD patients (mean: 336/microl+/-161 vs. controls 1008/microl+/-424, p=0.0001), with EBNA1-specific CD4 T cell responses being also low, but highly variable. Moreover, low absolute CD4 T cell counts (<230/microl) were associated with an elevated EBV load (>1000 copies/microg DNA). We conclude that SOT recipients with PTLD have an inadequate functional EBV-specific T cell response. Our data suggest that the frequency and function of circulating EBV-specific CD8 T cells are dependent on absolute CD4 T cell counts. Further studies are needed to verify if a low absolute CD4 T cell count presents a risk factor for the development of PTLD in SOT recipients.