Immortalized chromaffin cells disimmortalized with Cre/lox site-directed recombination for use in cell therapy for pain after partial nerve injury

To prepare immortalized adrenal chromaffin cells for eventual clinical use, the immortalizing oncogene must be removed. We have utilized a Cre-mediated excision of a loxP-flanked Tag sequence to test whether immortalized chromaffin cells could be disimmortalized by this method. Cultures of embryonic rat adrenal cells were immortalized with the tsA-TN retroviral vector encoding the loxP-flanked temperature-sensitive allele of SV40 large T antigen (tsA-TN) and a positive/negative neo/HSV-TK sequence for selection with either G418 or gancyclovir, respectively. These cells were then infected with the 1710-CrePR1 bicistronic retroviral vector coding for a form of Cre modulatable by the synthetic steroid RU486. These immortalized loxTsTag/CrePR1/RAD cells expressed immunoreactivities (ir) for all the catecholamine enzymes: tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH), and phenylethanolamine-N-methyltransferase (PNMT). After initial incubation at 37 degrees C with RU486 for 3 days, followed by the addition of gancyclovir for 7 days, Tag-ir was not detectable in most of the surviving chromaffin cells, compared to 100% expression in immortalized loxTsTag/CreR1/RAD cells not treated with RU486 and gancyclovir. The expression of TH, DbetaH, and PNMT was increased after disimmortalization and the ability of disimmortalized cells to synthesize norepinephrine was also significantly increased compared to immortalized cells. When both types of chromaffin cells were transplanted in a model of neuropathic pain and partial nerve injury, both cell grafts were equally able to reverse the behavioral hypersensitivity induced by the injury. The use of Cre/lox site-directed disimmortalization of chromaffin cells that are able to deliver neuroactive molecules offers a novel approach to cell therapy.     

Prognostic value of concordant seizure focus localizing data in the selection of temporal lobectomy candidates

This study was performed to test the hypotheses that (a) resection of the temporal lobe epileptic focus, amenable to noninvasive as opposed to invasive localization, is associated with superior seizure outcome and (b) that quadruple (versus lesser degrees of) concordance of seizure focus localizing data predicts superior seizure-free outcome. Eighty-three patients underwent invasive (subdural-EEG) and/or noninvasive (video/scalp-EEG, SPECT, PET, MRI, neuropsychological testing) evaluation. All patients underwent anterior temporal lobectomy and amygdalohippocampectomy (ATL/AH) and seizure outcome was assessed at minimum one-year follow-up. At 34.8 +/- 2.5 months following ATL/AH, outcome was superior for patients in whom the seizure focus was amenable to noninvasive compared to invasive localization (80% versus 40% seizure-free, X2 = 14.03, p < 0.05). Seizure outcome was superior for patients with quadruple, compared to all lesser degrees of, concordance of seizure focus localizing data (85% versus 51% seizure-free, X2 = 7.34, p < 0.05). Post-ATL/AH, seizure outcome is superior in patients (1) harboring an epileptic focus amenable to noninvasive localization and (2) with quadruple concordance of seizure focus localizing data. These findings support the development of temporal lobectomy selection criteria including up to four invasive and/or noninvasive concordant seizure focus localizing techniques.     

In vivo spectroscopy: a novel approach for simultaneously estimating nitric oxide and hemodynamic parameters in the rat brain

Nitric oxide (NO) is a versatile molecule involved in a wide range of biological processes. Under physiological conditions, NO reacts with oxyhemoglobin (OxyHb) to form methemoglobin (MetHb) at a very high rate. Microdialysis studies have used hemoglobin solutions as a trapping method to quantify NO in vivo. The methodology described here uses the microcapillary network with endogenous OxyHb instead of microdialysis probe with exogenous OxyHb for monitoring MetHb as an indirect index of NO levels by in vivo spectroscopy using optical fibers. This new method has been validated in rat cerebral cortex by the infusion of NO or well-known drug-induced changes in NO concentration (NMDA agonists and a NO-synthase inhibitor) and by comparing results with simultaneous voltammetric recordings. Results indicate that this spectroscopy technique is able to record large increases in MetHb levels and to detect reductions of its basal levels. In addition, data show that similar changes and kinetics can be observed with both techniques. Thus, intravascular MetHb can be used as an indirect index of NO levels. It is proposed that in vivo spectroscopy may be a useful tool to gain insight into the roles of NO in hemodynamic parameters and in other physiological processes such as the regulation of the mitochondrial respiratory chain.     

Zinc sulfate inhibits the enterohepatic cycling of unconjugated bilirubin in subjects with Gilbert's syndrome

We have previously observed that UCB binds to ZnSO4 in vitro, and suppressed the biliary bilirubin secretion in the hamster. The aim of this study was designed to investigate whether Zn salts might inhibit the enterohepatic cycling of UCB in subjects with Gilbert s syndrome. Fifteen patients with Gilbert s syndrome and 5 normal healthy volunteers were included in this study according to the following criteria: fasting hyperbilirubinemia, no hemolysis, and free of any medication. Patients were randomly assigned to receive acute o chronic treatment. Subjects treated in acute form and normal healthy volunteers were treated with 40 mg of ZnSO4 in a single dose, where as patients treated in chronic form received 100 mg ZnSO4 in a single dose daily for 7 days. The serum UCB levels (mg/dL) decreased from 2.64 +/- 1.04 to 2.02 +/- 0.87 (p < 0.001) and 1.8 +/- 0.36 to 1.48 +/- 0.32 (p < 0.005) in subjects treated in acute an chronic form respectively, but not in the control group. Whereas, the serum Zn levels (mg/dL) increased from 96.3 +/- 16.8 to 118.8 +/- 19. 5, (p < 0.01) and from 117.6 +/- 8.5 to 130.7 +/- 6.6 (p < 0.03) in subjects treated in acute an chronic form and also in subjects in the control group (98.0 +/- 7.3 to 128.0 +/- 21.9) p < 0.03. This study showed that acute and chronic oral administration of ZnSO4 decreased serum UCB levels significantly in subjects with Gilbert s syndrome. Most likely by the inhibition of the "normal" enterohepatic cycling of UCB.     

Citrobacter koseri in scalp folliculitis

Gram-negative folliculitis, an uncommon condition, is most often seen in older patients who have acne and who either have received prolonged courses of antibiotic therapy or have used antibacterial cleansers that selectively inhibit gram-positive organisms. Citrobacter infections are uncommon, and dermatologists seldom encounter them. In the past, these infections occurred in hospitals, particularly in neonatal intensive care units. Bacteremias also occur in elderly or immunocompromised patients. In this article, we present a case of Citrobacter koseri scalp folliculitis in an otherwise healthy patient.