Sodium-dependent vitamin C transporter 2 (SVCT2) is necessary for the uptake of L-ascorbic acid into Schwann cells.

Ascorbic acid has been shown to be an essential component for in vitro myelination and to improve the clinical and pathological phenotype of a mouse model of Charcot-Marie-tooth disease 1A. The mechanism of ascorbic acid uptake into peripheral nerves, however, has not been addressed so far. Hence, we studied the expression and activity of sodium-dependent vitamin C transporters 1 and 2 (SVCT1 and 2) in the peripheral nervous system. Using immunohistochemistry, immunoblotting, and reverse transcription PCR, we could show that SVCT1 and 2 were differentially expressed in myelinated peripheral nerve fibers and Schwann cell (SC) cultures. SVCT1 was expressed at very low levels confined to the axons, whereas SVCT2 was highly expressed both in the axons and in the SCs. SVCT2 was localized particularly in SC compartments of uncompacted myelin. Uptake assays using (14)C-labeled ascorbic acid showed transport of ascorbic acid into SC cultures. Ascorbic acid transport was dependent on the concentration of sodium, magnesium, and calcium in the extracellular medium. Treatment with the flavonoid phloretin, a known inhibitor of SVCT1 and 2, and specific RNA interference with SVCT2 caused significant reductions in ascorbic acid uptake into SCs. Phloretin-inhibited uptake of ascorbic acid was further shown in freshly dissected, cell-culture-naïve rat sciatic nerves. These results provide evidence for the first time that uptake of ascorbic acid in the peripheral nervous system is crucially dependent on the expression and activity of SVCT2.

TIMP-3: a novel target for glucocorticoid signaling at the blood-brain barrier.

Glucocorticoids (GCs) are used in the treatment of neuroinflammatory diseases such as multiple sclerosis. Several studies have demonstrated the beneficial effect of GCs on the balance between matrix metalloproteinases (MMPs) and their endogenous inhibitors, the TIMPs (tissue inhibitors of metalloproteinases). We could demonstrate that all four known TIMPs are present at the blood-brain barrier (BBB) endothelium. Hydrocortisone (HC) selectively upregulates TIMP-3 while TIMP-1, TIMP-2 and TIMP-4 were downregulated on the mRNA-level. This effect could be completely reversed by the glucocorticoid receptor inhibitor mifepristone (Mife). On the protein-level all TIMPs could be detected in the apical supernatants whereas in the isolated extracellular matrix (ECM) only TIMP-3 was found. The application of HC led to a strong enrichment of TIMP-3 in the ECM. Our findings demonstrate that HC directly targets TIMP-3 at the BBB assuming a protective role against matrix disruption and thus to guarantee the barrier integrity.

Expression of melanocyte differentiation antigens and ki-67 in nodal nevi and comparison of ki-67 expression with metastatic melanoma.

Nodal nevi represent a potential diagnostic pitfall in the analysis of lymph nodes. They may be confused with metastatic melanoma or carcinoma. Although several morphologic guidelines exist for the recognition of nodal nevi, on occasion immunohistochemical studies may be helpful for diagnosis, especially when melanocytes extend into the lymph node parenchyma. To learn more about the immunohistochemical profile of nodal nevi we examined 15 nodal nevi for the expression of S-100 protein, gp100 (HMB-45), Melan-A/MART-1 (A103), and tyrosinase (T311), and we studied the expression of Ki-67 (MIB-1) in nodal nevi and 40 melanoma metastases (35 lymph node and five cutaneous metastases). All nodal nevi were homogeneously immunoreactive for S-100 protein, tyrosinase, and Melan-A/MART-1. Two nodal nevi were focally positive for gp100. Fourteen of 15 nodal nevi were completely negative for Ki-67. One large cellular nodal nevus showed nuclear labeling in <0.2% of melanocytes. All metastases showed MIB-1 labeling. However, the percentage of labeled tumor cells varied widely, ranging from 2% to 80%. These results demonstrate that MIB-1 and HMB-45 are helpful reagents for the distinction of nodal nevi from melanoma. Immunohistochemistry for S-100 protein, Melan-A/MART-1, or tyrosinase facilitates the recognition of melanocytes but does not distinguish between nodal nevus and metastatic melanoma.

Sentinel lymph node biopsy in patients with diagnostically controversial spitzoid melanocytic tumors.

Melanomas can be difficult to diagnose histologically if they deviate in their growth pattern or cytology only minimally from a nevus. On occasion, even experts on melanocytic lesions may not reach a consensus on whether a lesion is a benign but unusual nevus or a malignant melanoma mimicking a nevus. This diagnostic dilemma is particularly well known for the distinction of Spitz nevus from melanoma. Diagnostic uncertainty and disagreement among consultant pathologists lead to confusion about the prognosis and clinical management of patients. In this study we present the clinical and pathologic findings of 10 patients with diagnostically controversial melanocytic tumors, who underwent sentinel lymph node biopsy. In all of these cases, the diagnostic controversy among experts was between Spitz nevus and melanoma. Seven patients were female, and three were male, ranging in age from 7 to 46 years (mean 21 years). Histologic examination of the sentinel lymph nodes revealed tumor deposits in the lymph node parenchyma in 5 of 10 patients. Among patients with positive sentinel lymph nodes, two had satellite nodules and one showed additional tumor deposits in three nonsentinel regional lymph nodes. All patients are alive and free of disease with a follow-up of 10-54 months (mean 34 months). Our study illustrates the role of a sentinel lymph node biopsy in the evaluation of patients with diagnostically controversial melanocytic tumors. Although the presence of metastatic tumor deposits in the sentinel lymph node supports the diagnosis of malignant melanoma, further studies are needed to determine the prognostic significance of the sentinel lymph node findings in such patients.

Tyrosine phosphatase inhibition induces loss of blood-brain barrier integrity by matrix metalloproteinase-dependent and -independent pathways.

Tight junctions between endothelial cells of brain capillaries form the structural basis of the blood-brain barrier (BBB), which controls the exchange of molecules between blood and CNS. Regulation of cellular barrier permeability is a vital and complex process involving intracellular signalling and rearrangement of tight junction proteins. We have analysed the impact of tyrosine phosphatase inhibition on tight junction proteins and endothelial barrier integrity in a primary cell culture model based on porcine brain capillary endothelial cells (PBCEC) that closely mimics the BBB in vitro. The tyrosine phosphatase inhibitor phenylarsine oxide (PAO) induced increased matrix metalloproteinase (MMP) activity, which was paralleled by severe disruption of cell-cell contacts and proteolysis of the tight junction protein occludin. ZO-1 and claudin-5 were not affected. Under these conditions, the transendothelial electrical resistance (TEER) was markedly reduced. PAO-induced occludin proteolysis could be prevented by different MMP inhibitors. Pervanadate (PV) reduced the TEER similar to PAO, but did not increase MMP activity. Cell-cell contacts of PV-treated cells appeared unaffected, and occludin proteolysis did not occur. Our results suggest that tyrosine phosphatase inhibition can influence barrier properties independent of, but also correlated to MMPs. Evidence is given for a role of MMPs in endothelial tight junction regulation at the BBB in particular and probably at tight junctions (TJs) in general.

Primary malignant melanoma of the oesophagus: a clinical and pathological study with emphasis on the immunophenotype of the tumours for melanocyte differentiation markers and cancer/testis antigens.

SUMMARY: Primary malignant melanomas of the oesophageal squamous mucosa are exceedingly rare. We present here the clinical and pathological findings of 10 patients (mean age 64 years) with primary oesophageal melanoma, with emphasis on the immunophenotype of the tumours. The majority of melanomas were located in the mid to distal oesophagus and were large (mean tumour size at the time of diagnosis 6.2 cm; mean depth of invasion 1.86 cm). All but two of the melanomas were associated with an extensive in situ component. Half of the tumours were amelanotic. The histological spectrum was wide, including appearances mimicking lymphoma, poorly differentiated adenocarcinoma or sarcoma. Immunohistochemical studies were performed on six tumours using monoclonal antibodies (MAb) to S100 protein, tyrosinase (MAb T311), Melan-A (MAb A103), and gp100 (MAb HMB-45), as well as antibodies to five cancer/testis (CT) antigens (MAb CT7-33 to CT7/MAGE-C1, MAb ESO121 to NY-ESO-1, MAb 57B to MAGE-A4, MAb MA454 to MAGE-A1, and MAb M3H67 to MAGE-A3). Seven patients had metastatic disease at the time of presentation. All but one patient underwent resection of the tumour with negative surgical margins. Survival was poor, with a mean survival of 19.8 months. One patient, however, whose tumour was limited to the submucosa, is still alive 108 months post-oesophagectomy. All six melanomas examined by immunohistochemistry were positive for all the melanocyte differentiation markers tested. In addition, they were all positive for CT antigens, with MA454 being the most commonly found, suggesting that CT antigens may be a promising immunotherapeutic target for oesophageal melanomas.

Identification of novel substrates and structure-activity relationship of cellular uptake mediated by human organic cation transporters 1 and 2.

Recently the clinical importance of human organic cation transporters 1 (hOCT1/SLC22A1) and 2 (hOCT2/SLC22A2) in drug disposition, for example, clearance, toxicity, and drug-drug interactions, have been highlighted [Annu. Rev. Pharmacol. Toxicol. 2012, 52, 249-273; Nat. Rev. Drug Discovery 2010, 9 (3), 215-236]. Consequently, there is an extensive need for experimental assessment of structure-transport relationships as well as tools to predict drug uptake by these transporters in ADMET (absorption, distribution, metabolism, excretion, toxicity) investigations. In the present study, we developed a robust assay for screening unlabeled compound uptake by hOCT1 and hOCT2 using transfected HEK293 cells. For the first time, an extensive data set comprising uptake of 354 compounds is presented. As expected, there was a large overlap in substrate specificity between the two organic cation transporters. However, several compounds selectively taken up by either hOCT1 or hOCT2 were identified. In particular, a chemical series of phenylthiophenecarboxamide ureas was identified as selective hOCT1 substrates. Moreover, the drivers for transport differed: molecular volume was the most important determinant of hOCT1 substrates, whereas H-bonding parameters like polar surface area (PSA) dominated for hOCT2.

Scintillation proximity assay for measuring uptake by the human drug transporters hOCT1, hOAT3, and hOATP1B1.

Increasing evidence suggests a key role of transport proteins in the pharmacokinetics of drugs. Within the solute carrier (SLC) family, various organic cation transporters (OCTs), organic anion transporters (OATs), and organic anion transporting polypeptides (OATPs) that interact with drug molecules have been identified. Traditionally, cellular uptake assays require multiple steps and provide low experimental throughput. We here demonstrate the use of a scintillation proximity approach to detect substrate uptake by human drug transporters in real time. HEK293 cells stably transfected with hOCT1, hOATP1B1, or hOAT3 were grown directly in Cytostar-T scintillating microplates. Confluent cell monolayers were incubated with 14C- or 3H-labeled transporter substrates. Cellular uptake brings the radioisotopes into proximity with the scintillation plate base. The resulting light emission signals were recorded on-line in a microplate scintillation counter. Results show time- and concentration-dependent uptake of 14C-tetraethylammonium, 3H-methylphenylpyridinium (HEK-hOCT1), 3H-estradiol-17beta-D-glucuronide (HEK-hOATP1B1), and 3H-estrone-3-sulfate (HEK-hOAT3), while no respective uptake was detected in empty vector-transfected cells. Km of 14C-tetraethylammonium and 3H-estrone-3-sulfate uptake and hOAT3 inhibition by ibuprofen and furosemide were similar to conventional dish uptake studies. The scintillation proximity approach is high throughput, amenable to automation and allows for identification of SLC transporter substrates and inhibitors in a convenient and reliable fashion, suggesting its broad applicability in drug discovery.

Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies.

This review brings you up-to-date with the hepatocyte research on: 1) in vitro-in vivo correlations of metabolism and clearance; 2) CYP enzyme induction, regulation, and cross-talk using human hepatocytes and hepatocyte-like cell lines; 3) the function and regulation of hepatic transporters and models used to elucidate their role in drug clearance; 4) mechanisms and examples of idiosyncratic and intrinsic hepatotoxicity; and 5) alternative cell systems to primary human hepatocytes. We also report pharmaceutical perspectives of these topics and compare methods and interpretations for the drug development process.

Congenital pauci-melanotic cellular blue nevus.

Unusual or atypical melanocytic nevi can be confused with malignant melanoma. Two patients are presented here with a rare variant of melanocytic nevus. Both were men. One was 39 years old and sought medical attention after trauma of a "congenital mole". The other was 24 years old and presented with a history of a slowly growing lesion, which had been known since childhood. In both patients, the lesion occurred on the buttock. They were dermal and superficial subcutaneous nodules measuring 1.5 and 2.3 cm in greatest dimension, respectively. The tumors were composed of densely cellular fascicles of melanocytes arranged in a lobulated growth pattern. Rare nests of small epithelioid melanocytes were also seen. No melanin pigment was seen on hematoxylin and eosin-stained sections. Focal minimal pigment was noted by Fontana-Masson stain in one case. Involvement of numerous peripheral nerve trunks by fusiform melanocytes was a prominent feature. Rare mitotic figures were seen in melanocytes [1-2 mitoses per 50 high-power fields (HPF)]. The MIB-1 labeling index was low (less than 5% of the lesional cell population was immunopositive). Both tumors were excised with negative surgical margins. One patient underwent sentinel lymph node biopsy because there was controversy regarding the biologic potential of the lesion. No melanocytic tumor deposits were found in the lymph nodes. On clinical follow up of 11 years and 18 months after complete excision, both patients are alive and well with no evidence of recurrence. We regard these lesions as congenital monophasic and pauci-melanotic variants of cellular blue nevus. The nevi are presented here to enhance our knowledge of the morphologic spectrum of melanocytic tumors and to help avoid confusion with malignant melanoma.

Survivin expression in ovarian carcinoma: correlation with apoptotic markers and prognosis.

Survivin is a novel inhibitor of apoptosis commonly detected in tissues during fetal development and in cancer, but not usually in normal tissues. Expression of this protein may be of prognostic significance and therapeutically relevant in many cancers. We assessed survivin expression in ovarian carcinoma, correlating results with expression of other anti-apoptotic (bcl-2, bcl-x, mutant p53) and pro-apoptotic (bax) markers, with prognostic parameters, and prognosis. Paraffin-embedded sections of 49 ovarian carcinoma were immunostained for survivin, bcl-2, bcl-x, bax, and p53. Expression was evaluated in nuclei and cytoplasm, as intensity (0-3+), and percentage of positive cells was scored on a four-tiered system with <10% as negative. Frequency of survivin, bcl-2, bcl-x, bax, and p53 was 73.5%, 36.7%, 93.9%, 77.6%, and 60.4%, respectively. There was significant correlation between nuclear survivin expression and grade (P =.0014), histologic type (P =.0376), and mutant p53 (P =.0414). Survivin expression did not correlate with bcl-2, bcl-x, or bax expression, stage, or overall or disease-free survival. The majority (74%) of ovarian carcinoma show survivin expression, which correlates with poor prognostic parameters (high grade, histologic type, p53 mutation) but not with survival. Therapeutic targeting of survivin in ovarian carcinoma is a future possibility.

In vitro studies indicate that miquelianin (quercetin 3-O-beta-D-glucuronopyranoside) is able to reach the CNS from the small intestine.

Miquelianin (quercetin 3-O-beta-D-glucuronopyranoside) is one of the flavonoids of St. John's wort (Hypericum perforatum L.) whose antidepressant activity has been shown by the forced swimming test, an in vivo pharmacological model with rats. However, nothing is known about its ability to reach the CNS after oral administration. We examined the pathway of miquelianin from the small intestine to the central nervous system using three in vitro membrane barrier cell systems. In the Caco-2 cell line, miquelianin showed a higher uptake (1.93 +/- 0.9 pmol x min(-1) x cm(-2)) than hyperoside (quercetin 3-O-beta-D-galactopyranoside; 0.55 +/- 0.18 pmol x min(-1) x cm(-2)) and quercitrin (quercetin 3-O-alpha-L-rhamnopyranoside; 0.22 +/- 0.08 pmol x min(-1) x cm(-2)). The permeability coefficient of miquelianin (Pc = 0.4 +/- 0.19 x 10(-6) cm/sec) was in the range of orally available drugs assuming sufficient absorption from the small intestine. Uptake and permeability of the examined compounds was increased by the MRP-2 inhibitor MK-571 indicating a backwards transport by this membrane protein. Porcine cell cultures of brain capillary endothelial cells were used as a model of the blood-brain barrier (bbb) and epithelial cells of the plexus chorioidei as a model of the blood-CSF barrier (bcb). Results indicate no active transport in one direction. Although moderate, the permeability coefficients (bbb: Pc = 1.34 +/- 0.05 x 10(-6) cm/sec; bcb: Pc = 2.0 +/- 0.33 x 10(-6) cm/sec) indicate the ability of miquelianin to cross both barriers to finally reach the CNS.