Primary Culture System Research Articles

Anandamide restricts uterine stromal differentiation and is critical for complete decidualization

The major endocannabinoid, anandamide (AEA), is widely distributed in the body, especially in the reproductive tissues, where it is implicated in early pregnancy events, particularly during implantation period. Although AEA is synthesized in decidual cells and showed to induce apoptosis through CB1 receptor, its roles in decidualization remain to be elucidated. This study examined the effect of AEA in the progression of decidualization both in vitro and in vivo and explored the involvement of COX-2 in its action. To determine the function of AEA during this differentiation process, we employed a primary culture system in which undifferentiated stromal cells isolated from pregnant rat uterus undergo decidualization. AEA treatment markedly interfered with the differentiation program, as revealed by α2-macroglobulin (α2-MG) expression and alkaline phosphatase activity. Additionally, it was evaluated the effects of AEA in decidual establishment in the pseudopregnant rat model. The abundance of AEA in the uterine lumen disrupted the decidualization process accompanied by a decreased expression of COX-2 and VEGF. It was also observed that uterine lumen, which failed the progression of decidualization in response to AEA, also presented lower expression of NAPE-PLD and FAAH. Thus, the mechanisms by which AEA inhibits decidualization can be either via direct actions on stromal cell differentiation within the reproductive tract system or by the inhibition of COX-2 derived products and, consequently, the vascular remodeling required to proper decidualization. In addition, the previous observations showing that higher AEA levels in pre-implantation sites are hostile to blastocyst survival may result from problems in decidual cell reaction more than with implantation failure.

Protective Effect of Lycii Radicis Cortex against 6-Hydroxydopamine-Induced Dopaminergic Neuronal Cell Death

An oxidative defense system imbalance leads to overproduction of reactive oxygen species (ROS) and is implicated in the progression of neurodegenerative ailments such as Parkinson's disease (PD). In the present study, we examined the protective effects of the ethanolic extract of Lycii Radicis Cortex (LRCE) in an in vitro PD model induced by 6-hydroxydopamine (6-OHDA), which induces selective dopaminergic cell death through oxidative stress. LRCE resulted in significant protective effects in SH-SY5Y cells and showed strong radical scavenging effects. In addition, LRCE inhibited intracellular ROS and extracellular nitrite production and glutathione depletion induced by 6-OHDA. Furthermore, LRCE blocked the destabilization of the mitochondrial membrane potential and the activation of caspase-3. Moreover, dopaminergic neuronal protection of LRCE from 6-OHDA exposure was confirmed in the rat primary mesencephalic culture system. LRCE is therefore considered to exert beneficial effects on dopaminergic neurons, resulting to antiparkinsonian effects via antioxidant activities. Practical Applications The current findings suggest that owing to its effects on antioxidant activity, Lycii Radicis Cortex may be useful as an alternative therapy to prevent and treat neurodegeneration including dopaminergic neuron dysfunction.

Effects of Danggui‐shaoyao‐san on Neuronal Damage in Parkinson's Disease Models

Danggui-shaoyao-san (DSS) has long been used Korean multiherbal medicine for the treatment of gynecological and neuro-associated disease. Recent studies reported that DSS has neuroprotection by multiple bioactivities. In the present study, we evaluated the effect of DSS on the anti-inflammatory against neuronal damage in an in vitro and in vivo model of PD. In primary mesencephalic culture system, DSS attenuated the dopaminergic cell damage induced by 1-methyl-4-phenylpyridine toxicity, and it inhibited production of inflammatory factors such as tumor necrosis factor α, nitric oxide, and activation of microglial cells. Then, we confirmed the effect of DSS in a mouse PD model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In the pole test, DSS at 50 mg/kg/day for 5 days showed increase of motor activity showing shortened time to turn and locomotor activity compared with the MPTP only treated mice. In addition, DSS significantly protected nigrostriatal dopaminergic neuron from MPTP stress. Moreover, DSS showed inhibition of gliosis in the substantia nigra pars compacta. These results have therapeutic implications for DSS in the treatment of PD via anti-inflammatory effects. This study was supported by a grant of the Korean Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea. (B120029).

Optimisation of proteomic approaches to study the maternal interaction with gametes in sow's reproductive tract

The applications of 2DE and MS have been successfully used in many studies utilising different biological samples. The complex nature of cellular proteomes is a big challenge for proteomic technologies. Much effort has been applied to develop and improve the preparation techniques for proteomic samples to be able to detect the low abundant proteins. This is one of the major and unsolved challenges facing the proteomic analysis of biological samples. One partial remedy is to deplete the proteomic samples. In this study, we compared two techniques (acetone precipitation and commercial kit) for the cleaning and purification of oviductal and uterine horn secretory proteomes in primary cell culture system. The samples prepared from acetone precipitation and commercial kit 2-D clean up kit were compared by 2-dimentioanl electrophoresis. We found that no significant difference was observed in number of spots detected between the samples prepared by acetone precipitation technique to those prepared by commercial kit. Protein samples were run through strong cation exchange (SCX) liquid chromatography in order to fractionate samples of major proteins. Protein identification by mass spectrometry revealed a significant detection of low abundant proteins in comparing to high abundant proteins. In conclusion, acetone precipitation was found to be more efficient and cost effect technique. Depletion of proteomic samples from the most abundant protein species is strongly recommended to allow the mid and low abundant protein to be detected. A better resolution of the gels will be achieved by removing the major proteins such as albumin and immunoglobulin.

Symposia and Oral Abstracts

[i]In vitro[/i] myotubes derived from zebrafish myogenic precursor cells upregulate Pax−3 and −7 following starvation. The zebrafish[i] (Danio rerio)[/i] remains the teleost fish of choice for biological investigations due to the plethora of molecular tools available for use in this system. However, its somatic growth is not representative of other teleost fishes, most of which are able to grow throughout their lives (termed indeterminate growth) while the zebrafish possesses a rather limited growth pot protein turnover, with the balance between protein synthesis and degradation governing whether myofibers hypertrophy or atrophy. To better describe the potentially divergent mechanisms of skeletal muscle atrophy in this species, we developed a primary myotube culture system generated from isolated myogenic precursor cells (MPCs) from adult zebrafish. Using a media devoid of serum and amino acids, we induced the expression of many genes associated with autophagy. Interestingly, paired−box transcription factor (Pax3/7) expression increased concurrently with myocyte enhancing factor−2ca (Mef2c) upregulation when de novo myotubes were subjected to starvation. From these results, it suggests that zebrafish myotubes, cultured from primary myoblasts, can be induced to express early myogenic biomarkers using nutrient alterations.

Endocytotic Uptake of Zoledronic Acid by Tubular Cells May Explain Its Renal Effects in Cancer Patients Receiving High Doses of the Compound

Zoledronic acid, a highly potent nitrogen-containing bisphosphonate used for the treatment of pathological bone loss, is excreted unmetabolized via the kidney if not bound to the bone. In cancer patients receiving high doses of the compound renal excretion may be associated with acute tubular necrosis. The question of how zoledronic acid is internalized by renal tubular cells has not been answered until now. In the current work, using a primary human tubular cell culture system, the pathway of cellular uptake of zoledronic acid (fluorescently/radiolabeled) and its cytotoxicity were investigated. Previous studies in our laboratory have shown that this primary cell culture model consistently mimics the physiological characteristics of molecular uptake/transport of the epithelium in vivo. Zoledronic acid was found to be taken up by tubular cells via fluid-phase-endocytosis (from apical and basolateral side) as evidenced by its co-localization with dextran. Cellular uptake and the resulting intracellular level was twice as high from the apical side compared to the basolateral side. Furthermore, the intracellular zoledronic acid level was found to be dependent on the administered concentration and not saturable. Cytotoxic effects however, were only seen at higher administration doses and/or after longer incubation times. Although zoledronic acid is taken up by tubular cells, no net tubular transport could be measured. It is concluded that fluid-phase-endocytosis of zoledronic acid and cellular accumulation at high doses may be responsible for the acute tubular necrosis observed in some cancer patients receiving high doses of the compound.

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed “enteroids” when derived from small intestine and “colonoids” when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Establishment of human epithelial enteroids and colonoids from whole tissue and biopsy.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed “enteroids” when derived from small intestine and “colonoids” when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Using Xenopus tissue cultures for the study of myasthenia gravis pathogenesis

Myasthenia gravis (MG), the most common autoimmune disease of neuromuscular junction (NMJ), is heterogeneous in terms of pathophysiology, which is determined by the pathogenic antigen of autoantibodies targeting to synaptic proteins at the NMJs. Currently, patients suspected with MG are routinely screened for the presence of autoantibodies against acetylcholine receptor (AChR) or muscle-specific kinase (MuSK) using a cell-based assay (CBA) that involves the expression of target synaptic membrane protein in heterologous cell lines. However, some autoantibodies may only show reactivity for binding to densely clustered AChR in the physiological conformation, while AChR clustering is known to involve signaling events orchestrated by over a dozen of postsynaptic proteins. To improve the existing serological diagnosis of MG, this study explored the possibility of using the well-established Xenopus primary culture system as a novel CBA for MG. Here, by examining the pathogenic effects of four MG human plasma samples, we found that the samples from both seropositive and seronegative MG patients effectively induced the disassembly of aneural AChR clusters in cultured Xenopus muscle cells, as well as the nerve-induced AChR clusters in the nerve–muscle co-cultures. Importantly, the disassembly of AChR clusters was spatio-temporally correlated to the disappearance of actin depolymerizing factor (ADF)/cofilin, an actin regulator involved in AChR trafficking and clustering. Taken together, this study develops a reliable CBA using Xenopus primary cultures for screening the pathogenicity of human MG plasma samples, and providing a platform for investigating the pathogenic mechanisms underlying the endocytic trafficking and degradation of AChRs at NMJs in MG patients.

Regulators of autophagosome formation in Drosophila muscles.

Given the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells, including orthologs of the Rab regulators RabGap1 and Rab3Gap1. The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. Specifically, sustained proteasome inhibition inhibited autophagosome formation both in primary culture and larval skeletal muscle, even though autophagy normally acts to suppress ubiquitin aggregate formation in these tissues. In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function.

Trehalose attenuates the gait ataxia and gliosis of spinocerebellar ataxia type 17 mice.

Spinocerebellar ataxia type 17 (SCA17) is caused by CAG/CAA repeat expansion on the gene encoding a general transcription factor, TATA-box-binding protein (TBP). The CAG repeat expansion leads to the reduced solubility of polyglutamine TBP and induces aggregate formation. The TBP aggregation, mostly present in the cell nuclei, is distinct from that in most other neurodegenerative diseases, in which the aggregation is formed in cytosol or extracellular compartments. Trehalose is a disaccharide issued by the Food and Drug Administration with a Generally Recognized As Safe status. Lines of evidence suggest trehalose could prevent protein aggregate formation in several neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and Huntington's disease. In this study, we evaluated the therapeutic potential of trehalose on SCA17 using cerebellar primary and organotypic culture systems and a mouse model. Our results showed that TBP nuclear aggregation was significantly decreased in both the primary and slice cultures. Trehalose (4 %) was further supplied in the drinking water of SCA17 transgenic mice. We found both the gait behavior in the footprint analysis and motor coordination in the rotarod task were significantly improved in the trehalose-treated SCA17 mice. The cerebellar weight was increased and the astrocyte gliosis was reduced in SCA17 mice after trehalose treatment. These data suggest that trehalose could be a potential nontoxic treatment for SCA17.

Genome-wide association study follow-up identifies cyclin A2 as a regulator of the transition through cytokinesis during terminal erythropoiesis.

Genome-wide association studies (GWAS) hold tremendous promise to improve our understanding of human biology. Recent GWAS have revealed over 75 loci associated with erythroid traits, including the 4q27 locus that is associated with red blood cell size (mean corpuscular volume). The close linkage disequilibrium block at this locus harbors the CCNA2 gene that encodes cyclin A2. CCNA2 mRNA is highly expressed in human and murine erythroid progenitor cells and regulated by the essential erythroid transcription factor GATA1. To understand the role of cyclin A2 in erythropoiesis, we have reduced expression of this gene using short hairpin RNAs in a primary murine erythroid culture system. We demonstrate that cyclin A2 levels affect erythroid cell size by regulating the passage through cytokinesis during the final cell division of terminal erythropoiesis. Our study provides new insight into cell cycle regulation during terminal erythropoiesis and more generally illustrates the value of functional GWAS follow-up to gain mechanistic insight into hematopoiesis.

JSAP1/JIP3 and JLP regulate kinesin-1-dependent axonal transport to prevent neuronal degeneration.

Axonal transport is critical for neuronal development and function, and defective axonal transport has been implicated in neurodegenerative diseases. However, how axonal transport is regulated, or how defective transport leads to neuronal degeneration, remains unclear. Here, we report that c-Jun NH2-terminal kinase (JNK)/stress-activated protein kinase-associated protein 1 (JSAP1, also known as JNK-interacting protein 3 (JIP3)) and JNK-associated leucine zipper protein (JLP) are essential for postnatal brain development. Mice with a double-knockout (dKO) in Jsap1 and Jlp in the dorsal telencephalon developed progressive neuron loss. Using a primary neuron culture system with induced disruption of targeted genes, combined with gene rescue experiments, we show that JSAP1 and JLP regulate kinesin-1-dependent axonal transport with functional redundancy. We also show that the binding of JSAP1 and JLP to kinesin-1 heavy chain is crucial for interactions between kinesin-1 and microtubules. Furthermore, we describe a molecular mechanism by which defective kinesin-1-dependent axonal transport in Jsap1:Jlp dKO neurons causes axonal degeneration and subsequent neuronal death. JNK hyperactivation because of increased intra-axonal Ca(2+) in the Jsap1:Jlp dKO neurons was found to mediate both the axonal degeneration and neuronal death, in cooperation with the Ca(2+)-dependent protease calpain. Our results indicate that axonal JNK may relocate to the nucleus in a dynein-dependent manner, where it activates the transcription factor c-Jun, resulting in neuronal death. Taken together, our data establish JSAP1 and JLP as positive regulators of kinesin-1-dependent axonal transport, which prevents neuronal degeneration.

RIG-I detects HIV-1 infection and mediates type I interferon response in human macrophages from patients with HIV-1-associated neurocognitive disorders.

The aim of this study was to explore the precise role of retinoic acid-inducible gene-I (RIG-I) signaling in human immunodeficiency virus type 1 (HIV-1)-infected macrophages from patients with HIV-1-associated neurocognitive disorders (HAND). Postmortem brain tissues were collected from patients with HIV-1-associated dementia and were compared to samples collected from HIV serum-positive patients without dementia and HIV serum-negative patients. A human monocyte-derived macrophage (MDM) primary culture system was established to evaluate the expression of RIG-I in these samples. Knockdown of RIG-I pathways genes was employed and STAT1 expression and phosphorylation levels were examined to explore the molecular mechanisms of HAND. The expression of RIG-I in postmortem brain tissue from HAND patients was significantly higher than in patients who were HIV serum-positive without dementia or HIV serum-negative. Moreover, we demonstrated that HIV-1 infection could result in a significant increase in the level of RIG-I in human MDMs. Moreover, a correlation was found between the increase in RIG-I expression and STAT1 expression and phosphorylation. Accordingly, knockdown of RIG-I decreased the phosphorylation of STAT1 and downregulated interferon-related genes. These observations highlight the importance of RIG-I signaling in anti-HIV innate immunity in macrophages, which may be beneficial for the treatment of HIV and aid in the understanding of the neuropathogenesis of HAND.

Vitamin D can ameliorate chlorhexidine gluconate-induced peritoneal fibrosis and functional deterioration through the inhibition of epithelial-to-mesenchymal transition of mesothelial cells.

Background. Peritoneal dialysis (PD) can induce fibrosis and functional alterations in PD patients' peritoneal membranes, due to long-term unphysiological dialysate exposure, partially occurring via triggering of epithelial-to-mesenchymal transition (EMT) in peritoneal mesothelial cells (MCs). Vitamin D can ameliorate these negative effects; however, the mechanism remains unexplored. Therefore, we investigated its possible links to MCs EMT inhibition. Methods. Peritoneal fibrosis was established in Sprague-Dawley rats by chlorhexidine gluconate (CG) intraperitoneal injection for 21 days, with and without 1α,25(OH)2D3 treatment. Morphological and functional evaluation and western blot analysis of EMT marker were performed upon peritoneum tissue. In vitro study was also performed in a primary human peritoneal MC culture system; MCs were incubated with transforming growth factor-β1 (TGF-β1) in the absence or presence of 1α,25(OH)2D3. EMT marker expression, migration activities, and cytoskeleton redistribution of MCs were determined. Results. 1α,25(OH)2D3 ameliorated CG-induced morphological and functional deterioration in animal model, along with CG-induced upregulation of α-SMA and downregulation of E-cadherin expression. Meanwhile, 1α,25(OH)2D3 also ameliorated TGF-β1-induced decrease in E-cadherin expression, increase in Snai1 and α-SMA expression, intracellular F-actin redistribution, and migration activity in vitro. Conclusion. 1α,25(OH)2D3 can ameliorate CG-induced peritoneal fibrosis and attenuate functional deterioration through inhibiting MC EMT.

Successful Generation of Hepatitis B virus (HBV) Pseudotype Particle; A Versatile Tool for Identification of the HBV Receptor and Investigation of HBV Infectivity

It is near a half century since hepatitis B virus (HBV) was identified. HBV receptor molecules and the entry mechanism of HBV into hepatocytes have not been elucidated completely, though there are some reports on infection systems and on the receptor molecules. Thus, we still have not reached finding a real HBV receptor and there have been no useful and convenient infection system in vitro and in vivo for HBV, which makes it impossible for us to understand a precise HBV life cycle and HBV involved related diseases. An HBV infection system is really needed to explore ways and means of treatment of HBV related diseases based on evidence as well. Here, we designed and tried to generate an HBV pseudotype, which has a viral particle containing a retrovirus capsid and a genome inside surrounded by HBV membrane proteins. We proved successful generation of this pseudotype by immunoprecipitation with anti-HBVs antibodies and by CsCl density gradient ultracentrifugation, followed by RT-PCR targeting a retroviral gene, an EGFP gene in this case, respectively. Though our established system is constructed on growth dependent integration of retroviral genomes and thus was very hard to observe its infection in a primary human hepatocytes culture system, successful generation of the HBV pseudotype will make it possible for us to perform a biological assay to clone an HBV receptor based on infectivity and will facilitate its separation and identification

A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro

Background and aimsHelicobacter pylori is the causative agent of gastric diseases and the main risk factor in the development of gastric adenocarcinoma. In vitro studies with this bacterial pathogen largely...

TIP30 inhibits oligodendrocyte precursor cell differentiation via cytoplasmic sequestration of Olig1.

Differentiation of oligodendrocyte precursor cells (OPCs) is a prerequisite for both developmental myelination and adult remyelination in the central nervous system. The molecular mechanisms underlying OPC differentiation remain largely unknown. Here, we show that the thirty-kDa HIV-1 Tat interacting protein (TIP30) is a negative regulator in oligodendrocyte development. The TIP30(-/-) mice displayed an increased myelin protein level at postnatal day 14 and 21. By using a primary OPC culture system, we demonstrated that overexpression of TIP30 dramatically inhibited the stage progression of differentiating OPCs, while knockdown of TIP30 enhanced the differentiation of oligodendroglial cells remarkably. Moreover, overexpression of TIP30 was found to sequester the transcription factor Olig1 in the cytoplasm and weaken its nuclear translocation due to the interaction between TIP30 and Olig1, whereas knockdown of TIP30 led to more Olig1 localized in the nucleus in the initiation stage during OPC differentiation. In the cuprizone-induced demyelination model, there was a dramatic increase in NG2-expressing cells with nuclear location of Olig1 in the corpus callosum during remyelination. In contrast, within chronic demyelinated lesions in multiple sclerosis, TIP30 was abnormally expressed in NG2-expressing cells, and few nuclear Olig1 was observed in these cells. Taken together, our findings suggest that TIP30 plays a negative regulatory role in oligodendroglial differentiation.

A primary fish gill cell culture model to assess pharmaceutical uptake and efflux: Evidence for passive and facilitated transport

The gill is the principle site of xenobiotic transfer to and from the aqueous environment. To replace, refine or reduce (3Rs) the large numbers of fish used in in vivo uptake studies an effective in vitro screen is required that mimics the function of the teleost gill. This study uses a rainbow trout (Oncorhynchus mykiss) primary gill cell culture system grown on permeable inserts, which tolerates apical freshwater thus mimicking the intact organ, to assess the uptake and efflux of pharmaceuticals across the gill. Bidirectional transport studies in media of seven pharmaceuticals (propranolol, metoprolol, atenolol, formoterol, terbutaline, ranitidine and imipramine) showed they were transported transcellularly across the epithelium. However, studies conducted in water showed enhanced uptake of propranolol, ranitidine and imipramine. Concentration-equilibrated conditions without a concentration gradient suggested that a proportion of the uptake of propranolol and imipramine is via a carrier-mediated process. Further study using propranolol showed that its transport is pH-dependent and at very low environmentally relevant concentrations (ngL−1), transport deviated from linearity. At higher concentrations, passive uptake dominated. Known inhibitors of drug transport proteins; cimetidine, MK571, cyclosporine A and quinidine inhibited propranolol uptake, whilst amantadine and verapamil were without effect. Together this suggests the involvement of specific members of SLC and ABC drug transporter families in pharmaceutical transport.

Activation of Endogenous Anti-Inflammatory Mediator Cyclic AMP Attenuates Acute Pyelonephritis in Mice Induced by Uropathogenic Escherichia coli

The pathogenesis of pyelonephritis caused by uropathogenic Escherichia coli (UPEC) is not well understood. Here, we show that besides UPEC virulence, the severity of the host innate immune response and invasion of renal epithelial cells are important pathogenic factors. Activation of endogenous anti-inflammatory mediator cAMP significantly attenuated acute pyelonephritis in mice induced by UPEC. Administration of forskolin (a potent elevator of intracellular cAMP) reduced kidney infection (ie, bacterial load, tissue destruction); this was associated with attenuated local inflammation, as evidenced by the reduction of renal production of proinflammatory mediators, renal infiltration of inflammatory cells, and renal myeloperoxidase activity. In primary cell culture systems, forskolin not only down-regulated UPEC-stimulated production of proinflammatory mediators by renal tubular epithelial cells and inflammatory cells (eg, monocyte/macrophages) but also reduced bacterial internalization by renal tubular epithelial cells. Our findings clearly indicate that activation of endogenous anti-inflammatory mediator cAMP is beneficial for controlling UPEC-mediated acute pyelonephritis in mice. The beneficial effect can be explained at least in part by limiting excessive inflammatory responses through acting on both renal tubular epithelial cells and inflammatory cells and by inhibiting bacteria invasion of renal tubular epithelial cells.