Undiagnosed Primary Sjögren's Syndrome With Pleural Involvement.

Sjögren's syndrome (SS) is a systemic autoimmune disorder characterized by the lymphocytic infiltration of the exocrine glands, primarily the salivary and lacrimal glands, resulting in the dryness of the eyes and mouth. However, Sjögren's syndrome is not limited to glandular involvement, as it can also affect various other organ systems, leading to a wide range of extraglandular manifestations and delaying the diagnosis. In this scenario, a high level of suspicion is required.

Eosinophilic Variant of Granulomatosis With Polyangiitis.

Granulomatosis with polyangiitis (GPA) is a multisystemic necrotizing vasculitis with a special tropism to the respiratory tract and the kidneys. Although uncommon, GPA may be associated with hypereosinophilia and limited organ involvement. In these cases, American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) classification criteria may be insufficient to establish the diagnosis. We described a limited form of GPA, hypereosinophilia, and predominant skin involvement.

Establishment of a robust hepatitis C virus replicon cell line over-expressing P-glycoprotein that facilitates analysis of P-gp drug transporter effects on inhibitor antiviral activity.

P-glycoprotein (P-gp) is an active efflux pump affecting the pharmacokinetic (PK) profiles of drugs that are P-gp substrates. The Caco-2 bi-directional assay is widely used to identify drug-P-gp interactions in vitro. For molecules exhibiting non-classical drug properties however, ambiguous results limit its use in lead optimization. The goal of this study was to develop a robust cell-based assay system to directly measure the role of P-gp-driven efflux in reducing the potency of hepatitis C virus (HCV) replication inhibitors. Vinblastine (Vin) was employed to select for a Vin-resistant HCV replicon (313-11) from the parental cell line (377-2). The 313-11 cell line was >50-fold resistant to Vin and over-expressed P-gp, as determined by Western immunoblots. Increased expression of P-gp was mediated by up-regulation of the MDR1 transcript. The reduced potency of different classes of HCV replication inhibitors in the 313-11 P-gp cell line was restored in the presence of known P-gp inhibitors. Addition of the P-gp inhibitor, tariquidar, increased the uptake of a radiolabeled HCV replication inhibitor by 14-fold in the 313-11 replicon cell line. Finally, a positive correlation was demonstrated between potency in the 313-11 replicon and the bi-directional Caco-2 efflux ratio for a panel of HCV protease inhibitors. In conclusion, a robust P-gp HCV replicon cell-based assay has been developed to measure the effect of the P-gp efflux pump on the potency of different classes of HCV replication inhibitors. This system establishes a direct correlation between antiviral activity and the effect of P-gp efflux in a single cell line.

B-cell lymphomagenesis in autoimmune diseases: the missing links.

Patients with autoimmune diseases such as systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome have an increased risk of developing B-cell non-Hodgkin's lymphomas but the mechanisms behind this phenomenon remain unknown. By focusing on recent research reports we explore and discuss some of the proposed mechanisms that contribute to this link. The complexity is enormous and can involve genetic and environmental factors, chronic immune stimulation by antigens, and even the treatment for these autoimmune diseases. These mechanisms can be combined in different ways causing great variability in one's predisposition to lymphomagenesis. Knowing more about these pathways is urgent. The more we know about autoimmune diseases the better we can treat our patients effectively and the more we can prevent lymphomas from developing.

Cardiac 123I-MIBG scintigraphy and arrhythmic risk in left ventricular noncompaction.

Left ventricular noncompaction is an unusual but increasingly recognized cardiomyopathy, the etiology of which is still not definitely established. Clinical presentation includes a wide spectrum of scenarios, including heart failure, thromboembolism and malignant arrhythmias, with half of deaths occurring suddenly. Early detection of LVNC is therefore essential to prevent sudden cardiac death. To our knowledge, this is the first report of the presence of cardiac sympathetic nervous dysfunction, assessed by 123iodine-metaiodobenzylguanidine myocardial scintigraphy, in a patient with LVNC, preserved left ventricular systolic function and exercise-induced nonsustained ventricular tachycardia. This finding may be related to the increased arrhythmic risk observed in this cardiomyopathy, giving a new insight into the pathophysiology of LVNC.

Expression Profile of Drug and Nutrient Absorption Related Genes in Madin-Darby Canine Kidney (MDCK) Cells Grown under Differentiation Conditions.

The expression levels of genes involved in drug and nutrient absorption were evaluated in the Madin-Darby Canine Kidney (MDCK) in vitro drug absorption model. MDCK cells were grown on plastic surfaces (for 3 days) or on Transwell® membranes (for 3, 5, 7, and 9 days). The expression profile of genes including ABC transporters, SLC transporters, and cytochrome P450 (CYP) enzymes was determined using the Affymetrix® Canine GeneChip®. Expression of genes whose probe sets passed a stringent confirmation process was examined. Expression of a few transporter (MDR1, PEPT1 and PEPT2) genes in MDCK cells was confirmed by RT-PCR. The overall gene expression profile was strongly influenced by the type of support the cells were grown on. After 3 days of growth, expression of 28% of the genes was statistically different (1.5-fold cutoff, p < 0.05) between the cells grown on plastic and Transwell® membranes. When cells were differentiated on Transwell® membranes, large changes in gene expression profile were observed during the early stages, which then stabilized after 5-7 days. Only a small number of genes encoding drug absorption related SLC, ABC, and CYP were detected in MDCK cells, and most of them exhibited low hybridization signals. Results from this study provide valuable reference information on endogenous gene expression in MDCK cells that could assist in design of drug-transporter and/or drug-enzyme interaction studies, and help interpret the contributions of various transporters and metabolic enzymes in studies with MDCK cells.

[Myocardial viability assessment]. / Avaliação da viabilidade miocárdica.

The prognosis for patients with chronic coronary artery disease and severe left ventricular dysfunction is poor, despite advances in different therapies. The assessment of myocardial viability has become an important aspect of the diagnosis, prognosis and management of patients with ischemic cardiomyopathy. Patients with left ventricular dysfunction, with a substantial amount of severely ischemic myocardium are at highest risk, and are likely to benefit from coronary revascularization. Patients with predominantly scar tissue should be treated medically. Multiple imaging techniques have been developed to assess viable and nonviable myocardium by evaluating perfusion, cell membrane integrity, glucose metabolism, fibrosis and contractile reserve. PET FDG-F18, myocardial perfusion scintigraphy (with (201)Tl and (99m)Tc), dobutamine stress echocardiography and more recently magnetic resonance have been extensively evaluated for assessment of viability and prediction of clinical outcome after coronary revascularization. In general, nuclear imaging techniques have a higher sensitivity for the detection of viability, whereas techniques evaluating contractile reserve have higher specificity (with lower sensitivity). Magnetic resonance has a high diagnostic accuracy for assessment of the transmural extent of myocardial scar tissue. The aim of this article is to review the role of Nuclear Medicine in assessing myocardial viability and risk stratification in patients with advanced left ventricular dysfunction, and to compare it with other imaging modalities.

[Metastatic calcification detected on bone scintigraphy]. / Calcificação metastática detectada em cintigrafia óssea.

PURPOSE: To show the value of bone scintigraphy in the diagnosis of metastatic calcification in end-stage renal insufficiency. MATERIAL AND METHODS: The authors present a fifty-one-year-old male with terminal renal insufficiency of hypertensive renovascular etiology, on hemodialysis for the last 10 years, referred for a bone scan because of osteoarticular complaints, and no other associated symptoms or findings on physical examination. RESULTS: Whole body bone scintigraphy images (HMDP-99mTc, 925MBq), showed increased uptake in the lower half of both lungs, the stomach and renal parenchyma, compatible with metastatic calcification. CONCLUSIONS: Bone scintigraphy, due to its high sensitivity, even in the detection of early changes, might be a useful instrument in the initial evaluation and follow-up of patients with high risk of developing metastatic calcification.

I-123-MIBG cardiac uptake imaging, in familial dilated cardiomyopathy.

UNLABELLED: The pathological significance of myocardial adrenergic activity in patients with heart failure is well documented. No previous study has assessed the usefulness of I123-metaiodobenzylguanidine (123I-MIBG) cardiac uptake imaging for the evaluation of familial dilated cardiomyopathy (DCM). OBJECTIVE: To evaluate cardiac adrenergic activity, using 123I-MIBG cardiac uptake imaging, in members of a genotyped family with DCM. METHODS: Clinical evaluation, 12-lead ECG, 2D echocardiogram, heart rate variability analysis by 24h Holter, plasma B-type natriuretic peptide (BNP) measurements and 123I-MIBG cardiac imaging were performed in all participants. Anterior projection planar images and single photon emission computed tomographies of the thorax were obtained 20 min and 4 hours after the intravenous administration of 370 MBq of 123I-MIBG (early and late images). Heart/mediastinal (H/M) ratio and myocardial washout (MW) rate were obtained based on the anterior planar images. In polar maps, segmental uptake of 123I-MIBG was evaluated using a 4-grade visual score: grade 1 - uptake > 75% of maximum myocardial uptake (MMU); grade 2 - uptake 51-75% of MMU; grade 3 - uptake 26-50% of MMU; grade 4 - uptake < or = 25% of MMU. RESULTS: Eleven adults were included: 4 with DCM, 4 with isolated left ventricular enlargement (LVE), and 3 with normal echocardiogram. Patients with DCM and LVE presented higher MW rates, lower H/M ratios and higher visual score grades than those with normal 2D echocardiograms. One patient with a normal echocardiogram but carrying the disease locus also presented an abnormal MIBG cardiac scintigram. CONCLUSION: Patients with the phenotypic expression of the disease (DCM and LVE) and even carriers of the DCM gene with normal echocardiograms may present an abnormal MIBG cardiac scintigram, probably reflecting cardiac adrenergic hyperactivity. If confirmed in larger numbers, this method may be useful for the evaluation of DCM families.

Peptide transporter substrate identification during permeability screening in drug discovery: comparison of transfected MDCK-hPepT1 cells to Caco-2 cells.

The purpose of this study was to investigate the utility of stably transfected MDCK-hPepT1 cells for identifying peptide transporter substrates in early drug discovery and compare the characteristics of this cell line with Caco-2 cells. MDCK-hPepT1, MDCK-mock, and Caco-2 cells grown to confluence on 24-well Transwell were used for this study. Expression levels of different transporter proteins (PepT1, PepT2, P-gp) in these cell lines were assessed by qRT-PCR. Permeability studies were conducted in parallel in all the cells with a diverse set of peptide substrates using the optimized experimental condition: 100 microM, apical pH 6.0, basolateral pH 7.4, 2 hr incubation at 37 degrees C. Permeability studies were also conducted with classical P-gp substrates (tested in bi-directional mode) and paracellularly absorbed probes to investigate the differences between the cell lines. As expected, MDCK-hPepT1 cells express significantly higher level of PepT1 mRNA compared to both Caco-2 and MDCK-mock cells. Efflux transporter, P-gp, was expressed adequately in all the cell lines. Permeability studies demonstrated that classical peptide substrates had significantly higher permeability in stably transfected MDCK-hPepT1 cells compared to MDCK-mock and Caco-2 cells. The transfected MDCK-hPepT1 cells were qualitatively similar to Caco-2 cells with respect to functional P-gp efflux activity and paracellular pore activity. Stably transfected MDCK-hPepT1 cells have been demonstrated as a viable alternative to Caco-2 cells for estimating the human absorption potential of peptide transporter substrates. These cells behave similar to Caco-2 cells with regards to P-gp efflux and paracellular pore activity but demonstrate greater predictability of absorption values for classical peptide substrates (for which Caco-2 cells under-estimate oral absorption).

Human PEPT1 pharmacophore distinguishes between dipeptide transport and binding.

The human intestinal oligopeptide transporter (PEPT1) facilitates the absorption of dipeptides, tripeptides, and many peptidomimetic drugs. In this study, a large number of peptides were selected to investigate the structural features required for PEPT1 transport. Binding affinity was determined in a Gly-Sar uptake inhibition assay, whereas functional transport was ranked in a membrane depolarization assay. Although most of the peptides tested could bind to PEPT1, not all were substrates. As expected, single amino acids and tetrapeptides could not bind to or be transported by PEPT1. Dipeptide transport was influenced by charge, hydrophobicity, size, and side chain flexibility. The extent of transport was variable, and unexpectedly, some dipeptides were not substrates of PEPT1. These included dipeptides with two positive charges or extreme bulk in either position 1 or 2. Our results identify key features required for PEPT1 transport in contrast to most previously described pharmacophores, which are based on the inhibition of transport of a known substrate.

pH-dependent dissolution in vitro and absorption in vivo of weakly basic drugs: development of a canine model.

PURPOSE: The aim of this research was to develop a pH-dependent canine absorption model for studying pH effect on both dissolution in vitro and pharmacokinetics in vivo using the weak bases ketoconazole and dipyridamole as model drugs. METHODS: Ketoconazole and dipyridamole pH-dependent dissolution profiles in vitro were determined by dissolution test at different pH values using USP apparatus II and an Opt-Diss Fiber Optic UV System. In vivo absorption studies for ketoconazole and dipyridamole were performed with crossover design in three groups of beagle dogs under control (no treatment), pentagastrin, and famotidine treatments. Ketoconazole and dipyridamole plasma concentrations were quantified by gradient high performance liquid chromatography mass spectroscopy (HPLC MS/MS). Pharmacokinetic parameters were determined from individual plasma concentration vs. time profiles. RESULTS: Ketoconazole and dipyridamole displayed pH-dependent dissolution. Increasing the pH of the dissolution medium from 1.2 to 6.8 reduced the extent of dissolution of ketoconazole and dipyridamole at 1 h by 96% and 92%, respectively. In vivo studies in dogs under control (no treatment), pentagastrin, and famotidine treatments show marked differences in systemic ketoconazole and dipyridamole exposure. Area under the concentration-time curve (AUC) increased more than 4-fold as compared to control group, whereas it increased nearly 30-fold for ketoconazole and 9-fold for dipyridamole with pentagastrin (gastric pH approximately 2-3) as compared to famotidine (gastric pH approximately 5-7.5) treatment. CONCLUSIONS: This work demonstrates a pH-dependent dissolution in vitro and absorption in vivo for the weak bases ketoconazole and dipyridamole independent of food effects. This model is useful to examine pH-dependent effects on oral drug absorption and for screening formulations to overcome the pH dependency.

Retinoic acid causes cell growth arrest and an increase in p27 in F9 wild type but not in F9 retinoic acid receptor beta2 knockout cells.

We have previously shown that an F9 teratocarcinoma retinoic acid receptor beta(2) (RARbeta(2)) knockout cell line exhibits no growth arrest in response to all-trans-retinoic acid (RA), whereas F9 wild type (Wt), F9 RARalpha(-/-), and F9 RARgamma(-/-) cell lines do growth arrest in response to RA. To examine the role of RARbeta(2) in growth inhibition, we analyzed the cell cycle regulatory proteins affected by RA in F9 Wt and F9 RARbeta(2)(-/-) cells. Flow microfluorimetry analyses revealed that RA treatment of F9 Wt cells greatly increased the percentage of cells in the G1/G0 phase of the cell cycle. In contrast, RA did not alter the cell cycle distribution profile of RARbeta(2)(-/-) cells. In F9 Wt cells, cyclin D1, D3, and cyclin E protein levels decreased, while cyclin D2 and p27 levels increased after RA treatment. Compared to the F9 Wt cells, the F9 RARbeta(2)(-/-) cells exhibited lower levels of cyclins D1, D2, D3, and E in the absence of RA, but did not exhibit further changes in the levels of these cell cycle regulators after RA addition. Since RA significantly increased the level of p27 protein (approximately 24-fold) in F9 Wt as compared to the F9 RARbeta(2)(-/-) cells, we chose to study p27 in greater detail. The p27 mRNA level and the rate of p27 protein synthesis were increased in RA-treated F9 Wt cells, but not in F9 RARbeta(2)(-/-) cells. Moreover, RA increased the half-life of p27 protein in F9 Wt cells. Reduced expression of RARbeta(2) is associated with the process of carcinogenesis and RARbeta(2) can mediate the growth arrest induced by RA in a variety of cancer cells. Using both genetic and molecular approaches, we have identified some of the molecular mechanisms, such as the large elevation of p27, through which RARbeta(2) mediates these growth inhibitory effects of RA in F9 cells.

A novel high-throughput pepT1 transporter assay differentiates between substrates and antagonists.

PepT1 is a transporter of proven pharmaceutical utility for enhancing oral absorption. A high-throughput, robust functional assay, capable of distinguishing PepT1 binders from substrates, allowing identification and/or prediction of drug candidate activation was developed. An MDCK epithelial cell line was transfected with rPepT1. The high level of stable rPepT1 expression that was achieved enabled development of a miniaturized PepT1 assay in a 96-well format, which could be scaled to 384 wells. The assay is based on measurement of membrane depolarization resulting from the cotransport of protons and PepT1 substrates. Membrane potential changes are tracked with a voltage-sensitive fluorescent indicator. Control (mock-transfected) cells are used to determine nonspecific membrane potential changes. A variety of fluorescent dyes were tested during initial assay design, including intracellular pH and membrane potential indicators. A membrane potential indicator was chosen because of its superior performance. Upon PepT1 activation with glycylsarcosine, dose-dependent membrane depolarization was observed with an EC50 of 0.49 mM. Maximum depolarization was dependent on the level of PepT1 expression. Testing of 38 known PepT1 substrates, binders, and nonbinders demonstrated that this assay accurately distinguished substrates from binders and from nonbinders. Initial validation of this novel assay indicates that it is sensitive and robust, and can distinguish between transporter substrates and antagonists. This important distinction has been previously achieved only with lower-throughput assays. This assay might also be used to determine substrate potency and establish a high-quality data set for PepT1 SAR modeling.

A novel hPepT1 stably transfected cell line: establishing a correlation between expression and function.

Stably transfected MDCK/hPepT1-V5&His clonal cell lines expressing varying levels of epitope-tagged hPepT1 protein were established to quantify the relationship between transgene hPepT1 expression levels and its functional kinetics in facilitating peptide and peptide-like drug uptake and transport in vitro. The hPepT1 sequence was amplified from Caco-2 cell mRNA, inserted into the pcDNA3.1 -V5&His TOPO plasmid, and transfected into MDCK cells. Transgene protein levels were quantified by Western Blot analysis utilizing a standard curve generated with a positive control protein containing a V5&His epitope. Three clones expressing different levels of the hPepT1 fusion protein (low, medium, and high) were selected for the functional characterization with [14C]Gly-Sar and [3H]carnosine. The MDCK/hPepT1 cells expressed a novel hPepT1/epitope tag protein with an apparent molecular mass of 110 kDa. The [14C]Gly-Sar uptake in the transfected cells was sodium-independent and pH-dependent, demonstrating enhanced uptake, the rate of which increased significantly from the weakly to strongly expressing hPepT1 MDCK/hPepT1 -V5&His clones as compared to the mock cell line at pH 6.0. The uptake and permeability of [14C]Gly-Sar and [3H]carnosine demonstrated a direct correlation between the hPepT1 level of expression, uptake, and transport capabilities. Molecular and functional characterization of the MDCK/hPepT1-V5&His cell line confirmed a directly proportional relationship between Vmax and Papp versus the molar levels of hPepT1 transgene expression. This stably transfected hPepT1 cell line may serve as a useful in vitro model for screening and quantifying peptide and peptide-like drug transport as a function of hPepT1 expression in drug discovery.

Identification and characterization of retinoic acid receptor beta2 target genes in F9 teratocarcinoma cells.

Retinoids, a group of natural and synthetic analogues of vitamin A (retinol), modulate the differentiation of many cell types. Retinoids are also used for the prevention and treatment of cancer. The actions of retinoids are generally mediated by the retinoic acid receptors (RARs alpha, beta, and gamma) and the retinoid X receptors (RXRs alpha, beta, and gamma). One of the RARs, RARbeta, is expressed at reduced levels in many human carcinomas, and F9 RARbeta(2)(-/-) cells do not growth arrest in response to RA. To determine if RARbeta(2) regulates the expression of a unique set of genes, through the use of subtractive hybridization and DNA array analysis, we have identified and characterized genes that are differentially expressed in F9 RARbeta(2)(-/-) teratocarcinoma cells. These genes, which encode transcription factors, cell surface signal transduction molecules, and metabolic enzymes, include c-myc, FOG1, GATA6, glutamate dehydrogenase, glutathione S-transferase homologue (p28), Foxq1, Hic5, Meis1a, Dab2, midkine, and the PDGF-alpha receptor. These genes are regulated specifically by RARbeta(2) in F9 wild-type (Wt) cells as indicated by their expression profiles in F9 RARbeta(2)(-/-) cells as compared to F9 Wt, RARalpha(-/-), or RARgamma(-/-) cells, and their responsiveness to specific retinoid receptor agonists. The basal expression levels of some of these genes, such as c-myc, are higher in the F9 RARbeta(2)(-/-) cells than in F9 Wt in the absence of exogenous retinoids, suggesting that RARbeta(2) can inhibit gene expression in the absence of a ligand. The RARbeta(2) target genes are transcriptionally activated by retinol, as well as RA, in F9 Wt cells. Because the lack of RARbeta(2) alters both the control of proliferation and differentiation in F9 cells, the genes that we have characterized may mediate key effects of RA, via RARbeta(2), on these processes.