Pretreatment of the cells with curcumin inhibited radiation-induced NF-κΒ activity and target protein expression. In vivo, the combination of curcumin and IR had synergistic antitumor activity against Rh30 and Rh41 ARMS xenografts. The greatest effect occurred when tumor-bearing mice were treated with curcumin prior to IR. Immunohistochemistry revealed that combination therapy significantly decreased tumor cell proliferation and endothelial cell count, and increased tumor cell apoptosis. Stable expression of the super-repressor, SR-IκBα, that blocks the classical NF-κB pathway, increased sensitivity to IR, while expression of SR-p100, that blocks the alternative pathway, did not. Our results demonstrate that curcumin can potentiate the antitumor activity of IR in ARMS xenografts by suppressing a classical NF-κΒ activation pathway induced by ionizing radiation. These data support testing of curcumin as a radiosensitizer for the clinical treatment of alveolar rhabdomyosarcoma.Ionizing radiation (IR) is an essential component of therapy for alveolar rhabdomyosarcoma. Nuclear factor-kappaB (NF-κΒ) transcription factors are upregulated by IR and have been implicated in radioresistance. We evaluated the ability of curcumin, a putative NF-κΒ inhibitor, and cells expressing genetic NF- κΒ inhibitors (IκBα and p100 super-repressor constructs) to function as a radiosensitizer. Ionizing radiation induced NF-κΒ activity in the ARMS cells The NF-κΒ protein complex has been linked to radioresistance in several cancers. In this study, we have demonstrated that inhibiting radiation-induced NF-κΒ activity by either pharmacologic (curcumin) or genetic (SR-IκBα) means significantly enhanced the efficacy of radiation therapy in the treatment of alveolar rhabdomyosarcoma cells and xenografts. These data suggest that preventing the radiation-induced activation of the NF-κΒ pathway is a promising way to improve the antitumor efficacy of ionizing radiation and warrants clinical trials. Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood Although ionizing radiation is a critical component of RMS therapy, it is associated with significant early and late complications. Early complications such as inflammation can occur within hours to several weeks after therapy. Changes in cell membrane permeability and histamine release subsequently lead to cell loss, causing mucositis and epidermal desquamation. Late effects of ionizing radiation include cognitive defects, hypothyroidism, musculoskeletal deformities, pulmonary fibrosis, cardiac dysfunction and second malignancies The nuclear factor-kappaB (NF-κΒ) family of transcription factors regulate the expression of genes involved in immune and inflammatory responses, developmental processes, and cellular proliferation and apoptosis Curcuma longa, commonly known as turmeric. Curcumin has been shown to suppress NF-κΒ activation and down-regulate expression of NF-κΒ gene targets in vitro by inhibiting induction of NF-κΒ activity Curcumin (diferuloylmethane), a yellow-colored polyphenol, is an active component of in vitro and in orthotopic alveolar rhabdomyosarcoma xenografts in mice.Despite advances in multi-modality therapy, many children with ARMS face a poor prognosis and so new treatment strategies are desperately needed. In this study, we tested the ability of curcumin to inhibit NF-κΒ activity and to act as a radiosensitizer. Our studies demonstrate that curcumin can potentiate the antitumor effects of ionizing radiation The human alveolar rhabdomyosarcoma-derived cell lines, Rh30 and Rh41, were established at St. Jude Children's Research Hospital, Memphis, TN by Dr. Peter Houghton . The handling of these two cell lines in this manner has been supported by the St. Jude Children's Research Hospital IRB. Cells that stably express the IκBα and p100 super-repressors were made by selecting blastocidin-resistant cell populations following transduction with retroviral vectors encoding either an Iκβα mutant (SR-IκBα), a p100 mutant (SR-p100), or empty vector (EV). The constructs encoding the IκBα and p100 mutants were kindly provided by Dr. Jiandong Li  and Dr. Shao-Cong Sun , respectively.A liposomal formulation of curcumin was prepared to improve the delivery of the drug. 1, 2-Dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1, 2-dimyristoylsn-glycero-3-[phosphor-rac- (1-glycerol)] (DMPG) were obtained from Avanti Polar Lipids . Curcumin , dimethylsulfoxide (DMSO), and tert-butanol were obtained from Sigma Chemical Company . Lyophilization of curcumin involved several steps. A 10∶1 total lipid to curcumin ratio (weight/weight) was used The effects of curcumin and IR on cell viability were determined by an Alamar Blue viability assay , according to the manufacturer's instructions. Cells were irradiated with a cesium-137 source and/or treated with liposomal curcumin. Alamar blue was added to treated cells and after 4 hours the absorbance was read on a Synergy 2 Multi-Mode Microplate Reader . These experiments were performed in triplicate.To analyze the effects of curcumin on apoptosis, cells were treated with 10 μmol of liposomal curcumin for 24 hours before irradiation. Cells were analyzed by flow cytometry for apoptosis (Annexin V staining) at 48 hours following irradiation.32P-end-labeled double-stranded NF-κΒ oligonucleotide containing a tandem repeat consensus sequence of 5′-AGT TGA GGG GAC TTT CCC AGG C-3′, and separated by electrophoresis on 4% polyacrylamide gels. Bands were visualized on PhosphorImager and quantified with ImageQuant 5.2 software .Nuclear extracts were prepared using the Affymetrix Nuclear Extraction Kit  and stored at −80°C. To determine NF-κΒ DNA binding activity, an electrophoretic mobility shift assay (EMSA) was performed on nuclear extracts following the protocol by Promega Gel Shift Assay Systems. In brief, nuclear extracts were incubated with a Antibodies for matrix metalloproteinase-9 (MMP-9), IκBα, and pIκBα were obtained from Santa Cruz Biotechnology, Santa Cruz, CA. Antibodies for bcl-2 and XIAP were obtained from Cell Signaling . Antibodies for Cox-2 were obtained from Cayman Chemical . Protein expression of MMP-9, Cox-2, bcl-2, XIAP, IκBα and pIκBα was determined by western blot analysis as previously described 2 x length x 0.5. Mice with size-matched Rh30 or Rh41 xenografts were placed into one of four groups approximately 4 weeks after tumor cell injection and then treated with empty liposome given intraperitoneally (IP), 50 mg/kg liposomal curcumin IP daily until sacrifice, 10 Gy IR, or combination therapy. A single dose of 10 Gy IR was administered using an Orthovoltage D3000 x-ray tube  All murine experiments were done in accordance with a protocol approved by the Institutional Animal Care and Use Committee of St. Jude Children's Research Hospital. Orthotopic intramuscular (IM) ARMS xenografts were established in male CB-17 severe combined immunodeficient mice , as previously described in vivo of Rh30 cell lines that express the SR-IκBα or SR-p100 super-repressor construct. Xenografts of Rh30-EV, Rh30 SR-IκBα, and Rh30 SR-p100 were established in the right calf of mice. For each xenograft, mice were placed into 2 groups of equivalent tumor burden to receive 10 Gy IR or to serve as controls.In addition, we examined the response in situ apoptosis detection kit  as previously described Formalin-fixed, paraffin-embedded, 4-μm thick tumor sections were stained with rat anti-mouse CD34  antibodies as previously described in vivo experiments, Bliss's independent joint action principle was used to define a synergy index (SI). Continuous variables are reported as mean ± SEM and were compared using an unpaired Student's t test with a p-value <.05 considered significant. Data were analyzed and graphed using Sigma Plot .Isobolograms were used to assess the in vitro synergy between curcumin and radiation To determine NF-κΒ DNA binding activity in Rh30 and Rh41 cells exposed to ionizing radiation (10 Gy), nuclear extracts were prepared at various times after irradiation and analyzed by electrophoretic mobility shift assay (EMSA). Both cell lines showed a time-dependent induction of NF-κΒ DNA binding activity, with activity evident within 1 hr after radiation exposure, and reaching a maximum activity at ∼2 hr . To furtThe p50:p65 complex of NF-κΒ is induced through activation of the classical NF-κΒ pathway, which involves IκBα serine phosphorylation, polyubiqitination and subsequent proteosomal degradation. Therefore, expression of a mutant IκBα construct, in which the inducible serine phosphorylation sites have been mutated to alanine, should block classical NF-κΒ activation. In the alternative pathway, activation of the NF-κΒ-inducing kinase leads to proteolysis of p100 and liberation of the p52 complexes. A mutant p100 construct, in which the inducible serine phosphorylation sites have been mutated to alanine, should block the activation of the alternative NF-κΒ pathway. To determine whether radiation-induced NF-κΒ activation in ARMS cells involved the classical or alternative pathway, EMSA was performed on nuclear extracts prepared from ARMS cells that expressed IκBα and p100 super-repressors, respectively. As shown in ARMS cells were treated with curcumin, a pharmacological inhibitor of NF-κΒ activity, to determine whether curcumin could abrogate the radiation-induced activation of NF-κΒ. Rh30 and Rh41 cells were pretreated with varying doses of liposomal curcumin  for varying lengths of time  prior to receiving a single dose of 10 Gy IR. Nuclear extracts were prepared 2 hr following radiation and assayed for NF-κΒ DNA binding activity by EMSA. As shown in 50 for inhibition of cell proliferation by curcumin was similar in both cell lines (17.5±1.92 µM for Rh30 to 19.3±1.89 µM for Rh41) , confirming that there was no loss in the potency of curcumin during liposomal formulation. Treatment with empty liposomes alone had no effect on the proliferation of the rhabdomyosarcoma cell lines (data not shown). IR inhibited proliferation of Rh30 and Rh41 cells, in a dose dependent manner as well. The IC50 doses varied from 18.3±1.3Gy for Rh30 to 18.9±1.2Gy for Rh41  . The antfor Rh41 To test the effects of combination therapy, cells were pretreated with varying doses of curcumin for 24 hr prior to exposure to varying doses of radiation and cell viability was assessed at 96 hr following curcumin administration. The potential synergy between curcumin and radiation, on cell viability was assessed by an isobologram based-approach .  prior to radiation exposure (10 Gy) and then analyzed by FACS for apoptosis by Annexin V-staining 48 hr after irradiation. The combination of curcumin and radiation significantly increased apoptosis compared to control in both cell lines. Apoptosis in control Rh30 cells was 4.6%±0.41 as compared to 8.67%±0.64 or 7.5%±0.3 in cells treated with radiation, or curcumin alone, respectively. In contrast apoptosis in Rh30 cells treated with a combination of radiation and curcumin was 19.5%±0.54 (p = .00003). A similar trend was observed in Rh41 cells. Apoptosis in control cells was 6.63%±0.66 as compared to 10.1%±1.6, or 9.9%±0.8 in cells treated with radiation or curcumin alone, while the combination of radiation and curcumin resulted in 22.9%±1.5 apoptosis (p = .0001) .Since the classical NF-κΒ pathway involves the degradation of IκB, we investigated whether radiation induced IκBα degradation in IR-treated rhabdomyosarcoma cells. Whole cell extracts were prepared from Rh30 and R41 cells at various times after radiation exposure (10 Gy). Western blot showed IR induced a decrease in cellular IκBα levels within 1 hr and IκBα levels returned to control levels by 4 hr . TreatmeNF-κΒ has been shown to regulate the expression of target genes involved in proliferation (cyclin D1), inflammation (COX-2), metastasis (MMP-9), and anti-apoptosis (XIAP and Bcl-2). We next performed western blot analysis to determine whether curcumin modulated these target proteins in rhabdomyosarcoma cells treated with IR. This analysis revealed that, while radiation induced the expression of cyclin D1, COX-2, MMP-9, XIAP, and Bcl-2, curcumin treatment suppressed their induction by radiation .To further demonstrate the functional role of the radiation-induced canonical NF-κΒ pathway, the effect of IR on proliferation of Rh30 and Rh41cells expressing the IκBα super-repressor was analyzed. The Rh30 and Rh41 cells expressing the IκBα super-repressor were significantly more sensitive to the antiproliferative effects of 10 Gy IR when compared to the control (empty-vector expressing) cells and cells expressing the p100 super-repressor. The proliferation of irradiated Rh30 EV and Rh30 SR-p100 cell lines was 85.73±1.38% and 84.62±0.9% of control non-irradiated cells, respectively, as compared to 71.48±0.9% in the irradiated Rh30 cells expressing SR- IκBα (p = 0.0002). The proliferation of irradiated Rh41 EV and Rh41 SR-p100 cell lines was 81.66±0.53% and 80.63±0.44% of control nonirradiated cells, respectively, as compared to 68.93±1.7% in the Rh41 SR- IκBα cell line (p = 0.0002) .The effects of the NF-κΒ super-repressors were also analyzed by Annexin V-staining 48 hr after irradiation. Both Rh30 and Rh41 SR-IκBα cells had a significantly increased number of apoptotic cells compared to empty vector after exposure to 10 Gy radiation. The Rh30 EV and SR-p100 cell line had 10.87±0.9% and 11.73±1.5% Annexin V positive cells, respectively, as compared to 24.56±2.2% for the IκBα super-repressor cell line. The Rh41 EV and SR-p100 cell line had 11.66±0.9% and 12.8±0.5% Annexin V positive cells, respectively, as compared to 21.3±1.5% Annexin V positive cells in the IκBα super-repressor cell line (p = 0.0007) .in vivo. An orthotopic model of Rh30 and Rh41 ARMS was established in the right calf of CB-17 severe combined immunodeficient mice. After ∼1 month, mice with established xenografts were size-matched by tumor volume into 4 groups (n = 9/group). Mice received lyophilized vehicle IP daily, 50 mg/kg lyophilized curcumin IP daily, a single dose of 10 Gy IR, or combination therapy. A single dose of 10 Gy IR was given on day 7 of curcumin treatment for the group receiving combination therapy. Curcumin treatment was continued until mice were sacrificed. Following 3 weeks of treatment, the combination of curcumin and radiation had synergistic antitumor activity in the Rh30 xenografts . The combination group had a tumor burden of 201.8±19.33 mm3 compared to 786±44.4 mm3, 679±51.46 mm3, and 1033.4±89.71 mm3 for the curcumin, radiation and control groups, respectively. Combination therapy in the Rh41 xenografts was also synergistic . The combination group had a tumor burden of 275.1±55.9 mm3 compared to 927.4±98.7 mm3, 904.9±94.2 mm3and 1221.7±232.4 mm3 for the curcumin, radiation and control groups, respectively (We next determined whether curcumin sensitized rhabdomyosarcoma to IR ectively .3 compared to 242±27.5 mm3 for the group that was pretreated with curcumin before radiation (p = 0.0017). In the Rh41 cell line, the group that received radiation before curcumin administration had a tumor volume of 756±92.4 mm3 compared to 227±26.4 mm3 for the group that was pretreated with curcumin before radiation (p = 0.0003) (We next confirmed the importance of the timing of curcumin administration on the effectiveness of radiation exposure. Rh30 and Rh41 tumors were established and mice were size-matched into 3 groups (n = 5). Mice received lyophilized vehicle IP daily, 10 Gy radiation followed immediately by 50 mg/kg liposomal curcumin IP daily, and 10 Gy radiation 7 days after the start of liposomal curcumin. Curcumin was continued until the animals were sacrificed. In both rhabdomyosarcoma xenografts, the effect of the timing of curcumin treatment prior to irradiation was found to be critical. In the Rh30 cell line, the group that received radiation before curcumin administration had a tumor volume of 620±68.5 mm 0.0003) . These rin vitro studies, we performed western blot analysis for IκBα from tumor tissues harvested post-IR to determine the mechanism whereby curcumin inhibits NF-κΒ activation in vivo. This analysis showed that in both rhabdomyosarcoma xenografts, radiation-induced NF-κΒ activation was mediated by IκBα degradation . In the Rh41 cell line, the combination therapy group had a proliferation index of 66.8±0.98% compared to 81.44±1.7%, 80.77±1.6% and 86.61±0.84% for the curcumin group, radiation group and control group, respectively (p<0.0001) .Next, we examined tumor cell apoptosis by TUNEL staining. In both cell lines, the greatest increase in apoptotic cells was found with combination therapy. In the Rh30 cell line the combination group had 18.46±1.8 apoptotic cells/1000 cells compared to 12.15±1.1, 12.14±1.4 and 8.98±1.1 apoptotic cells/1000 cells for the curcumin group, radiation group and the control groups, respectively (p<0.0001). In the RH41 cell line the combination group had 17.11±1.1 apoptotic cells/1000 cells compared to 11.67±1.09, 10.90±.85 and 9.52±0.5 apoptotic cells/1000 cells for the curcumin group, radiation group and the control group (p<0.0004) .We next examined tumor microvessel density by assessing CD34 immunohistochemistry. The combination therapy resulted in the greatest decrease in tumor microvessel density in both rhabdomyosarcoma xenografts. In the Rh30 cell line, combination therapy group had 20,022±2,225 CD34 pixels/hpf compared to 33,363±2,055, 37,481±2,514 and 41,245±3,225 CD34 pixels/hpf for the curcumin group, radiation group and the control group, respectively (p<0.0001) In the Rh41 cell line, the combination therapy group had 14,353±1,740 CD34 pixels/hpf compared to 34,827±2,357, 38,994±2,955 and 46,121±4,393 CD34 pixels/hpf for the curcumin group, radiation group and the control group, respectively (p<0.0007) .Next, we established orthotopic Rh30 EV, SR-p100 and SR-IκBα xenografts. Approximately 1 month after injection, mice bearing tumors of each cell line were size-matched into 2 groups, one group was subjected to IR (n = 10) while the other served as control. Five mice in each group were sacrificed 4 hr after exposure to 10 Gy radiation to confirm suppression of NF-κΒ activity. In the control tumors and tumors derived from the p100 super-repressor expressing cells exposed to IR, there was an increase in the NF-κΒ activity, specifically in complexes containing the p50 and p65 subunits. There was no increase in the NF-κΒ activity upon radiation exposure in tumors derived from SR-IκBα expressing cells , confirm3 vs. 1,019±61 mm3, p<0.0003). Tumors with empty vector treated with IR had a mean tumor volume of 967±64.6 mm3 that was only 27% less than the mean tumor volume of 1,331±133 mm3 in mice that did not receive radiation. Similarly, tumors derived from the p100-SR expressing cells when treated with IR, had a volume of 790±133mm3, which was only 30% less than the mean tumor volume of 1,125±109 mm3 in mice not receiving IR  and dose (10 Gy) dependent manner. Supershift assays performed with antibodies directed to specific NF-κΒ subunits showed that the p50 and p65 subunits comprised the radiation-induced NF-κΒ complex. We confirmed that IR-mediated induction of NF-κΒ activity occurred via the classical pathway in experiments with enforced expression of super repressor constructs which showed abrogation of radiation-mediated NF-κΒ activation in cell lines transduced with an SR-IκBα mutant  but not an SR-p100  mutant.Treatment with curcumin, a naturally occurring inhibitor of NF-κΒ activity, prior to the delivery of ionizing radiation, inhibited the upregulation of NF-κΒ activity in the ARMS cell lines and xenografts in response to IR, as demonstrated by EMSA. This in turn led to increased IR-mediated tumor cell apoptosis and antitumor efficacy. Activation of the classical NF-κΒ pathway is normally achieved by phosphorylation and subsequent degradation of IκBα, a cytoplasmic protein that binds and inactivates NF-κΒ by preventing its translocation into the nucleus. In our study of ARMS xenografts treated with curcumin prior to ionizing radiation, curcumin prevented the degradation of IκBα, by inhibiting the formation of the activated form of IκBα (phospho-IκBα).in vitro. We did not perform time course experiments in vivo in our xenograft model but did find improved antitumor activity when mice were treated with curcumin for one week prior to the delivery of ionizing radiation as compared to when both were given concomitantly.The timing of curcumin administration was critical to its radiosensitization effect as its ability to effect NF-κΒ inhibition prior to the administration of ionizing radiation was essential. We found maximal induction of NF-κΒ activation two hours after the delivery of ionizing radiation to ARMS cells In addition to demonstrating that blocking activation of NF-κΒ by curcumin resulted in IR-mediated induction of NF-κΒ-responsive genes involved in cell proliferation (cyclin D1) and survival (XIAP and bcl-2), we showed that the combination of curcumin and IR caused a profound decrease in microvessel density in rhabdomyosarcoma xenografts. Thus, the mechanism of synergy between curcumin and IR is likely to be multifactorial and involves direct tumor cell cytotoxicity, inhibition of angiogenesis as well as radiosensitization.in vivo study with this formulation showed it to be well-tolerated and therapeutic doses should be easily achieved.One of the disadvantages of curcumin for use in the clinical setting is its limited solubility. We have overcome this problem by using liposomal curcumin. Our This is the first study, to our knowledge, that demonstrates the adjuvant antitumor activity of curcumin in vivo against rhabdomyosarcoma tumors treated with radiation therapy. In addition, we have shown that this in vivo radiosensitization is due to the abrogation of the increase in the NF-κB activity that occurs in these solid tumors in response to ionizing radiation via the classical pathway. We further confirmed these findings through suppressing NF-κB activation by genetic means. These data suggest that preventing the radiation-induced activation of the NF-κΒ pathway is a promising way to improve the antitumor efficacy of adjuvant ionizing radiation and warrants study in clinical trials.Figure S1Effects of curcumin, radiation and combination therapy on rhabdomyosarcoma tumor cell proliferation. (A) Curcumin treatment resulted in a dose-dependent decrease in proliferation in both Rh30 and Rh41 cell lines. IC50 concentrations determined at 96 hr after treatment varied from 17.5 µM±1.92 for Rh30 and 19.3 µM±1.89 for Rh41 (B) Radiation therapy resulted in a dose-dependent decrease in proliferation in both the Rh30 and Rh41 cell lines. IC50 doses determined at 72 hr after treatment varied from 18.3±1.3Gy for Rh30 to 18.9±1.2Gy for Rh41. (C) The effects of combination therapy was synergistic in both cell lines. The Rh30 treatment resulted in a positive α non-additivity parameter of 5.54 with a 95% confidence interval . The Rh41 treatment resulted in an α of 5.86 with a 95% confidence interval .(TIF)Click here for additional data file.Figure S2Effects of curcumin, radiation and combination therapy on rhabdomyosarcoma apoptosis. Cells were treated with 10 µM liposomal curcumin (24 hr) prior to radiation exposure (10 Gy) and then analyzed for apoptosis by Annexin V staining. The combination therapy of curcumin and irradiation significantly increased apoptosis compared to control in both the Rh30 and Rh41 cell lines .(TIF)Click here for additional data file.Figure S3Curcumin inhibits radiation-induced upregulation of NF-κΒ dependent genes. Western blot analysis revealed that radiation induced NF-κΒ dependent genes , which was suppressed by curcumin pretreatment in Rh30 and Rh41.(TIF)Click here for additional data file.Figure S4IκBα super-repression sensitizes rhabdomyosarcoma to radiation in vitro. (A) Rhabdomyosarcoma cell lines expressing the IκBα super-repressor were more sensitive to radiation compared to the cell lines expressing empty vector or p100 super-repressor. (B) The Rh30 and Rh41 SR-IκBα cell lines showed a significant increase in Annexin V positive cells compared to the cell lines expressing empty vector and SR-p100.(TIF)Click here for additional data file.Figure S5Curcumin enhances the effect of radiation on tumor cell proliferation, apoptosis and angiogenesis. Ki67, TUNEL, CD34 immunohistochemistry staining was used to analyze Rh30 and Rh41 tumors for tumor cell proliferation, apoptosis and angiogenesis, respectively (A) Combination therapy resulted in a significant decrease in tumor cell proliferation , shown is representative Ki-67 staining (400X) (B) Combination therapy resulted in a significant increase in the TUNEL positive cells compared to control , shown is representative TUNEL staining (400X) (C) Combination therapy resulted in a significant decrease in tumor microvessel density compared to control tumors , shown is representative CD34 staining (400X).(TIF)Click here for additional data file.


Macrophages and fibroblasts are two major players in tissue repair and fibrosis. Despite the relevance of macrophages and fibroblasts in tissue homeostasis, remarkably little is known whether macrophages are able to influence the properties of fibroblasts. Here we investigated the role of paracrine factors secreted by classically activated (M1) and alternatively activated (M2) human macrophages on human dermal fibroblasts (HDFs).HDFs stimulated with paracrine factors from M1 macrophages showed a 10 to > 100-fold increase in the expression of the inflammatory cytokines IL6, CCL2 and CCL7 and the matrix metalloproteinases MMP1 and MMP3. This indicates that factors produced by M1 macrophages induce a fibroblast phenotype with pro-inflammatory and extracellular matrix (ECM) degrading properties. HDFs stimulated with paracrine factors secreted by M2 macrophages displayed an increased proliferation rate. Interestingly, the M1-activated pro-inflammatory fibroblasts downregulated, after exposure to paracrine factors produced by M2 macrophages or non-conditioned media, the inflammatory markers as well as MMPs and upregulated their collagen production.Paracrine factors of M1 or M2 polarized macrophages induced different phenotypes of HDFs and the HDF phenotypes can in turn be reversed, pointing to a high dynamic plasticity of fibroblasts in the different phases of tissue repair. In wound healing and fibrosis, a variety of processes are crucial, such as inflammation, cell proliferation, cell migration and extracellular matrix (ECM) remodeling. Two major cellular players in these processes are macrophages and fibroblasts -4. DurinThe other important cell type in wound healing and fibrosis, macrophages, exist as resident tissue-specific macrophages, or are derived from circulating blood monocytes that undergo diapedesis and subsequently differentiate into macrophages. Macrophages display various activation states. The two opposite activation states are known as classically activated (M1) and alternatively activated (M2) macrophages ,11. The In the inflammatory phase of wound healing, invading macrophages are pro-inflammatory (M1) and secrete several cytokines and chemokines, like chemokine (C-C motif) ligand 2 (CCL2) (monocyte chemotactic protein-1), CCL7 (monocyte chemotactic protein-3) and interleukin 6 (IL6). These cytokines/chemokines play a crucial role in wound healing and are involved in fibrogenesis ,15,18-21vice versa, in vitro[In vivo, there are indications that re-polarization of macrophages also occurs, as shown in a mouse model for atherosclerosis [It has been shown that macrophages show a high dynamic plasticity. Macrophages can change, depending on the stimulus in the micro-environment, their secretion pattern of cytokines and chemokines several times -26. For  in vitro. In vivoclerosis  and in aclerosis . This maDespite the relevance of macrophages and fibroblasts in tissue homeostasis, remarkably little is known whether the different types of human primary macrophages are able to influence directly the properties of human primary fibroblasts. Most of the data found in literature have generally been generated with cell lines -32 or prPrimary human macrophages responded to LPS/IFNG or IL4/IL13, resulting in M1 or M2 polarization, respectively. M1 polarized macrophages adopted a “dendritic”-like morphology with large filopodia while M2 polarized macrophages showed a rounded and/or spindle-shaped morphology, which was comparable with the morphology of unstimulated macrophages , a high expression of CD68, which is a general marker for macrophages. M1 macrophages had a lower CD68 expression than M2 polarized or unstimulated macrophages , IL6 and CCL2 compared to M2 polarized and unstimulated macrophages  and mannose receptor, C type 1 (MRC1) compared to M1 polarized or unstimulated macrophages. CCL18 tended to be upregulated in IL4/IL13 stimulated macrophages while interleukin 1 receptor, type II (IL1R2), which acts as a decoy receptor for the type I interleukin 1, showed a higher expression in IL4/IL13 and unstimulated macrophages than M1 polarized macrophages , a marker for myofibroblast formation, is upregulated at gene expression level by fibroblasts stimulated with CM of unstimulated macrophages compared to CM of M1 stimulated macrophages after 48 h, 72 h and 144 h. Fibroblasts stimulated with CM of M2 macrophages showed an upregulation of ACTA2 compared to fibroblasts stimulated with CM of M1 macrophages after 144 h Figure A. No difOn protein level no ACTA2 was seen in fibroblasts after 144 h of stimulation with the three different CM. This was in contrast to TGFB1 stimulated fibroblasts (myofibroblasts), which showed ACTA2 protein expression after 144 h Figure B. TGFB1 Together, these results indicate that CM from M1, M2 or unstimulated macrophages did not result in the differentiation of fibroblasts into myofibroblasts.After 72 h, fibroblast cell numbers were similar in all conditions, but increased exclusively after stimulation with CM of M2 macrophages after 144 h Figure A. NucleaThe results indicate that CM from M2 macrophages induced proliferation of fibroblasts.ECM deposition by fibroblasts is an important process in wound healing and fibrosis. Two major collagens produced in these processes are collagen type I (COL1A1) and collagen type III (COL3A1). COL1A1 gene expression in fibroblasts was reduced after stimulation with CM of M1 macrophages compared to CM of M2 and unstimulated macrophages after 144 h Figure A. CM of After 72 h, no difference in collagen type I deposition was seen after the different stimulations. However, less collagen type I protein deposition was seen by fibroblasts stimulated with CM of M1 macrophages compared to the other conditions after 144 h . No differences were seen in TAGLN gene expression between the three conditions (data not shown).COL1A1 gene expression was upregulated after the switch of CM compared to fibroblasts stimulated with M1 macrophages CM at 144 h Figure C. This gThe results indicate that the effects of factors produced by M1 macrophages on HDFs diminish once HFDs are not exposed to these factors anymore (i.e. if HDFs are exposed to M2-CM or non-CM).in vitro are widely used, but it should be realized that the macrophage phenotype in wounds likely exhibit a more complex phenotype in (certain stages of) wound healing [Macrophages play important roles in wound repair processes. Macrophages are phenotypically highly plastic, and their polarization state depends on the micro-environment present in the wounded area. The M1 and M2 polarization states are opposite activation states of a continuum. Protocols to induce M1 and M2 macrophages  healing -39. NeveIn this study we investigated the influence of secreted factors (conditioned medium) of M1 or M2 macrophages on dermal fibroblasts. Simultaneously, the influence of secreted factors of M1 macrophages followed by stimulation with secreted factors of M2 macrophages was investigated. In addition, we used conditioned medium from unstimulated macrophages. These unstimulated macrophages have a “M2-like” phenotype, which is probably caused by stimulating monocytes with macrophage colony-stimulating factor (M-CSF), a step that is necessary to induce differentiation of monocytes towards macrophages ,41. Despet al.[in vitro model with murine primary cells and cell lines, that fibroblasts produce pro-inflammatory cytokines and chemokines after stimulation with conditioned medium of LPS-stimulated macrophages and in a co-culture system with direct cell-cell contact. Other studies [In general, macrophages that invade the tissue in the inflammatory phase of wound healing adopt a M1 phenotype. In our model, the secreted factors from M1 macrophages influences the properties of dermal fibroblasts already within 24 h, changing the phenotype into a pro-inflammatory state. This indicates that fibroblasts, under the direction of paracrine signals of M1 macrophages, contribute to a pro-inflammatory environment by secreting cytokines and chemokines  in the inflammatory phase of wound healing. This is in accordance with data shown by Holt et al.. These a studies ,32,33 shin vivo the fibroblasts are able to prolong the inflammation state in wound healing by itself or by attracting more pro-inflammatory cells.MMPs are capable of regulating chemokine activity and ECM degradation in tissue repair ,43. MMPsFibroblasts exposed to conditioned medium from M2 macrophages showed little response. Only a slight increase was seen in the expression of ACTA2, but this did not resulted in myofibroblast formation. Furthermore, an increase in cell proliferation was seen, which was in accordance with previous findings ,23,31,44In wound repair it is thought that M2 macrophages are responsible for reversing the inflammatory response, thereby initiating the healing process. Interestingly, in this study we show that fibroblasts with an inflammatory phenotype (initiated by stimulation with secreted factors of M1 macrophages) can be reversed to an anti-inflammatory phenotype with secreted factors of M2 macrophages or non-CM. In these fibroblasts, the previously upregulated pro-inflammatory cytokines, chemokines, and MMPs were completely downregulated after stimulation with paracrine signals from M2 macrophages or non-CM. Thus, although paracrine factors of M2 macrophages have relatively little effect on unstimulated fibroblasts, they can have a major effect on fibroblasts with an inflammatory phenotype.In summary, we have shown that secreted factors from M1 macrophages gives rise to fibroblasts with a pro-inflammatory and ECM-degrading profile, while M2 macrophages induce fibroblast proliferation. The pro-inflammatory and ECM-degrading fibroblast can be reversed completely by secreted factors from M2 macrophages or non-CM. Therefore, not only macrophages, but also fibroblasts show a high dynamic plasticity in wound healing / tissue repair processes, a plasticity that seems to be regulated by the micro-environment.4Cl, 10 mM KHCO3, 0.1 mM EDTA (pH 7.4) and the suspension was centrifuged at 300 × g at 4°C for 10 min after which the supernatant was discarded and the pellet gently resuspended in isolation buffer. PBMCs were counted using a Coulter Counter .Human peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated from buffy coats  by density-gradient centrifugation using Lymphoprep  according to the manufacturer’s protocol. Briefly, blood was diluted three times with isolation buffer (pH 7.4) consisting of phosphate buffered saline (PBS) with 0.5% fetal bovine serum  and 2 mM EDTA . This mixture (30 ml) was layered over 20 ml of Lymphoprep and centrifuged at 800 × g for 30 min. Residual erythrocytes were lysed on ice (10 min) in 155 mM NH+ cells were isolated by immunomagnetic bead separation using CD14 Microbeads . Briefly, 1 × 107 PBMCs were labeled with 20 μl CD14 Microbeads and incubated on ice in 80 μl isolation buffer for 30 min. Cells were washed with isolation buffer and the suspension was centrifuged at 300 × g at 4°C for 10 min. The pellet was resuspended in degassed isolation buffer and the CD14+ cells were separated with an LS column  placed on a column adapter in a strong magnetic field. CD14+ cells bind to the column and after carefully washing with degassed isolation buffer and removal of the LS column from the magnet the CD14+ cells were flushed out from the column using a plunger. The CD14+ cells were counted with a Coulter Counter and after centrifugation at 300 × g for 10 min at 4°C gently resuspended in culture medium, consisting of X-VIVO-10 medium  supplemented with 2 mM l-glutamine , 1% penicillin/streptomycin  and 10 ng/ml recombinant human M-CSF .CD142 onto tissue culture polystyrene plates . Cells were cultured at 37°C under 5% CO2. Cells were refed at day 3 and non-attached cells were removed from culture at day 6.Immediately after isolation and counting, the cell suspension was plated with a density of 100,000 cells/ cmAt day 6, the adherent cells (macrophages) were washed and stimulated in culture medium (but without M-CSF), with either (1) 1 μg/ml LPS  + 10 ng/ml IFNG  , (2) 2 ng/ml IL4 + 2 ng/ml IL13  , or (3) no stimulation (control) at 37°C for 48 h. The polarization state of the macrophages was determined by quantitative RT-PCR (qRT-PCR). The cells were subsequently washed and cultured in X-VIVO-10 medium for 4 h (conditioned medium). After 4 h the CM from (1) M1 macrophages, (2) M2 macrophages and (3) unstimulated macrophages was collected and stored for further analyses at −20°C. The CM of the different conditions were used for stimulation of HDFs, the determination of CCL2 and CCL18 levels by means of enzyme-linked immunosorbent assays (ELISA) and the determination of cytokines with a multiplex bead immunoassay.2 in X-VIVO-10 medium containing 2 mM l-glutamine, 1% penicillin/streptomycin and 50 μg/ml l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate . The next day the X-VIVO medium was replaced by CM derived of M1, M2 or unstimulated macrophages, which was supplemented with l-ascorbic acid 2-phosphate sesquimagnesium salt hydrate. Passage 5 or 6 of HDFs were used for stimulations with CM from macrophages. The CM was refreshed every day and the stimulated HDFs were characterized at 24 h, 48 h, 72 h and 144 h by morphology, qRT-PCR and after 24 h, 72 h and 144 h by immunofluorescent stainings. The deposition of the extracellular matrix protein collagen type I was determined at 72 h and 144 h. After 24 h and 48 h, CM of stimulated HDFs was collected and stored for further analysis at −20°C. Prior to collection of the CM, the stimulated HDFs were washed and cultured in X-VIVO-10 medium for 4 h. CCL2, CCL7, IL6, MMP1, MMP2 and MMP3 secretion by HDFs was determined by ELISA. All culture conditions were carried out at 37°C under 5% CO2.Primary HDFs  were seeded onto TCPS overnight with a density of 15,000 cells/cmHDFs were cultured as described above. After overnight seeding in X-VIVO-10 medium the medium was replaced by CM of M1 macrophages for 24 h or 48 h, with refreshment of the CM after 24 h. After 24 h or 48 h the medium was replaced by CM of M2 macrophages or by X-VIVO-10 medium (non-CM) for another 48 h or 96 h, respectively ; the CM or non-CM were refreshed every day. The HDFs were characterized by qRT-PCR.Total RNA was isolated from the cells using the RNeasy Kit  in accordance to the manufacturer’s protocol. RNA concentration and purity were determined by UV spectrophotometry . For qRT-PCR analysis, total RNA was reverse transcribed using the First Strand cDNA synthesis kit  in accordance to the manufacturer’s protocol. Quantification of gene expression was performed using qRT-PCR analysis in a final reaction volume of 10 μl, consisting of 1× SYBR Green Supermix , 6 μM forward primer, 6 μM reverse primer Table  and 5 ng2O2 and stopped with 1 M H2SO4. The absorbance was measured using a microplate reader . The detection limit for MMP2, MMP1, MMP3, CCL2, IL6, CCL7 and CCL18 was 312 pg/ml, 78 pg/ml, 15.6 pg/ml, 7.8 pg/ml, 4.7 pg/ml and 3.9 pg/ml, respectively.Determination of CCL2, CCL7, CCL18, IL6, MMP1, MMP2 and MMP3 protein levels were measured using DuoSet® ELISA Development kit  in accordance to manufacturer’s protocol. Briefly, 96 wells plates  were coated with Capture Antibody and incubated overnight at room temperature (RT). After incubation the plates were washed with 0.05% Tween-20  in PBS and blocked with 1% bovine serum albumin (BSA)  in PBS for 1 h. After washing, the plates were incubated with diluted sample or matched standards for 2 h. The detection was performed using matched biotin conjugated antibodies followed by streptavidin-poly-horseradish peroxidase . The color reaction was performed with tetramethylbenzidine  in sodium acetate buffer, pH 6, containing HFactors that were secreted by M1, M2 and unstimulated macrophages were determined by a multiplex bead immunoassay in accordance to manufacturer’s protocol . Briefly, beads that have defined spectral properties and are conjugated to protein-specific capture antibodies were added to a 96 well filter plate. After washing, the plate was incubated with sample or matched standards for 2 h. The detection was performed using protein-specific biotinylated detector antibodies and streptavidin conjugated R-Phycoerythrin. The beads were analyzed with the Luminex-100 detection system .2, 0,1% Brij-35, pH 7.0 and 10 μM OmniMMP™ fluorogenic substrate . The fluorescent intensity was measured using a fluorescence plate reader  after 20 h of incubation at 37°C.MMP activity was determined in the CM of HDFs after 24 h of stimulation with CM derived of M1, M2 or unstimulated macrophages. The CM of the HDFs was mixed, in a black 96 flat bottom plate, with prewarmed assay buffer containing 0.1 M 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 20 mM CaClAfter 24 h and 144 h of culture, HDFs were washed twice with PBS and fixed in 2% paraformaldehyde (PFA) at RT for 10 min. Fixed cells were incubated with 0.5% Triton X-100  in PBS for 3 min at RT. After washing with PBS the cells were incubated with (1) mouse-anti-human ACTA2  (1:100) or (2) rabbit-anti-human MKI67  (1:500) diluted in PBS containing 1% BSA for 1 h at RT. After three washes with PBS, cells were incubated with biotinylated (1) goat-anti-mouse IgG2a-biotin  (1:100), or (2) goat-anti-rabbit-FITC  (1:100) diluted in PBS containing 2% normal human serum (NHS) for 30 min at room temperature. The cells were subsequently washed three times with PBS and incubated with streptavidine-CY3  (1:100) in PBS containing 1% BSA, 2% NHS and DAPI (1:5000) for 30 min. After three washes with PBS the slides were mounted in Citifluor  and examined by immunofluorescent microscopy using a Leica DMRA microscope equipped with a Leica DFC350FX digital camera and Leica Application Suite (LAS) software .After 72 h and 144 h of culture, HDFs were washed twice with PBS and fixed in 2% PFA at RT for 10 min. Fixed cells were incubated at RT with (1) mouse-anti-human collagen type I (COL I) (1:100)  diluted in PBS containing 1% BSA for 1h. The HDFs were washed three times with PBS, followed by incubation with goat-anti-mouse IgG1-biotin  (1:100) diluted in 1% BSA in PBS for 30 min. The cells were subsequently washed three times with PBS and incubated with streptavidine-CY3 (1:100) in PBS containing 1% BSA, 2% NHS and DAPI (1:5000) for 30 min. After three washes with PBS the slides were mounted in Citifluor and examined by immunofluorescent microscopy using a Leica DMRA microscope.2. After polymerization CM of M1, M2 or unstimulated macrophages was added. As control complete X-VIVO medium supplemented with 10 ng/ml TGFB1  was used. The CM and medium supplemented with TGFB1 was refreshed every day and the cells were cultured at 37°C under 5% CO2. After 5 days the gels were gently released and contractile force was analyzed by measuring the gel diameter at 8 h after release using a flatbed scanner  Data are expressed as the percentage of area compared to the initial gel area.Collagen gels were prepared by mixing X-VIVO-10 medium, 1 M NaOH, 10 × PBS, 0.2 M HEPES and collagen I . The final concentration was 5.2 mM NaOH, 1 × PBS, 2 mM HEPES, 2.4 mg/ml of collagen I in X-VIVO-10 medium. HDFs were added in a concentration of 200.000 cells/ml and 500 μl of this mixture was pipetted into a well of a 24-well culture plate. Polymerization of the solution occurred within 1h at 37°C under 5% COP < 0.05 were considered to be statistically significant.All data are represented as means ± standard error of the mean of at least three independent experiments and were analyzed by Graph-Pad Prism Version 5 for Macintosh  either by one-way ANOVA followed by Tukey’s post hoc analysis, or by two-way ANOVA followed by Bonferroni post hoc analysis. Values of Quantitative Reverse Transcription polymerase chain reaction; TAGLN: Transgelin; TCPS: Tissue culture polystyrene plates; TGFB1: Transforming growth factor beta 1; TIMP1: Tissue inhibitor of metalloproteinases −1; TLR4: Toll-like receptor 4; TMB: Tetramethylbenzidine; TNF: Tumor necrosis factor; YWHAZ: Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide.ACTA2: Alpha-actin-2; BSA: Bovine serum albumin; CCL: Chemokine (C-C motif) ligand; CLEC10A: C-type lectin domain family 10, member A; CM: Conditioned medium; COL1A1: Collagen type I; COL3A1: Collagen type III; CXCL: Chemokine (C-X-C motif) ligand; ECM: Extracellular matrix; FBS: Fetal bovine serum; FGF2: Fibroblast growth factor 2; HDFs: Human primary dermal fibroblasts; IFNG: Interferon gamma; IL: Interleukin; LPS: Lipopolysaccharides; M-CSF: Macrophage colony-stimulating factor; M1: Classically activated macrophages; M2: Alternatively activated macrophages; MKI67: Nuclear protein Ki-67; MMP: Matrix metalloproteinase; MRC1: Mannose receptor, C type 1; NHS: Normal human serum; PBMCs: Peripheral blood mononuclear cells; PBS: Phosphate buffered saline; PFA: Paraformaldehyde; qRT-PCR: The authors declare that they have no competing interests.DTAP designed research, performed experiments, analyzed data, interpreted data and wrote the manuscript. NAH designed research, performed experiments, interpreted data and edited the manuscript. MS contributed to preliminary experiments. JAK and SdR performed experiments and edited the manuscript. R.A.B. supervised the work, interpreted the data, and edited the manuscript. All authors read and approved the final manuscript.


In this report, we validated the trafficking and function of Oatp4c1 in polarized cell systems as well as its subcellular localization in rat kidney. Using several complementary biochemical, molecular and proteomic methods as well as antibodies amenable to immunohistochemistry, immunofluorescence, and immunobloting we investigated the expression pattern of Oatp4c1 in polarized cell systems and in the rat kidney. Collectively, these data demonstrate that rat Oatp4c1 traffics to the apical cell surface of polarized epithelium and localizes primarily in the proximal straight tubules, the S3 fraction of the nephron. Drug uptake studies in Oatp4c1-overexpressing cells demonstrated that Oatp4c1-mediated estrone-3-sulfate (E3S) uptake was pH-dependent and ATP-independent. These data definitively demonstrate the subcellular localization and histological location of Oatp4c1 and provide additional functional evidence that reconciles expression-function reports found in the literature.The organic anion transporting polypeptide 4c1 (Oatp4c1) was previously identified as a novel uptake transporter predominantly expressed at the basolateral membrane in the rat kidney proximal tubules. Its functional role was suggested to be a vectorial transport partner of an apically-expressed efflux transporter for the efficient translocation of physiological substrates into urine, some of which were suggested to be uremic toxins. However, our  Oatp family have been identified in the rat kidney proximal tubules. Oatp1a1 (previously: Oatp1) and Oatp1a3  are expressed at apical membranes 3) and thyroxine (T4)), cAMP, methotrexate (MTX) 3 uptake to 40% 4 uptake is significantly higher at extracellular pH 6.5 than pH 8.0 33 have mutual inhibition. Furthermore, the physiological role of OATP4C1, reportedly a basolateral uptake transporter, has been postulated to be coupling with P-glycoprotein, an apical efflux transporter, to facilitate the renal clearance of common substrates such as digoxin The kidney is responsible for homeostasis of endogenous and exogenous substances through tubular secretion and reabsorption, which in part is mediated by various membrane transporters, including the solute carrier family (SLC) and ATP-binding cassette (ABC) superfamily. Several studies have demonstrated that overlapping substrate specificity among uptake and efflux transporters is likely to accelerate the translocation of endogenous and exogenous substances across epithelial or endothelial barriers +/H+ exchanger regulatory factor 1 (NHERF1) expression, an adaptor protein, which determines the trafficking of MRP4 in vitro models and in rat tissues. We used a comprehensive approach including multiple antibodies amenable to immunohistochemistry, immunofluorescence, and immunobloting to probe the expression of rat Oatp4c1 in intact tissues and cells as well as biochemically separated and enriched apical and basolateral membranes isolated from polarized cells and renal proximal tubules. Furthermore, proteomic analysis was used to qualitatively validate the specificity of our antibody. Functional activity of Oatp4c1 in MDCKII-Oatp4c1 was probed with E3S. Collectively, our data provide evidence that is contrary to published work regarding the localization, polarity, and function of Oatp4c1.In addition to the kidney, there are limited data regarding the expression of OATP4C1. Microarray expression has shown that human SLCO4C1 is also expressed in the liver, lung, mammary gland, skin, neutrophils, peripheral leukocytes and mononuclear cells Tissues from animals were collected using procedures formally approved by the University of Kentucky Institutional Animal Care and Use Committee protocol# 2007-0228. All cell culture procedures and cell-line modifications were performed with approval from the Institutional Biosafety committee at the University of Kentucky under application B07-598.3H]-E3S (45.6 Ci/mmol) was from Perkin Elmer . GF120918 was a gift from GlaxoSmithKline . All other chemicals were purchased from Sigma-Aldrich  unless specified otherwise. All procedures involving the culture and manipulations of cell lines were approved by the University of Kentucky Biological Safety Committee., two carboxyl-terminus , and an extracellular loop [SPDFEARAGKC (PA1556)] previously reported by Mikkaichi and colleagues 20 primers according to the manufacturer’s protocol. Rat Slco4c1 cDNA was amplified using Phusion High-Fidelity PCR kit  with forward (5′- CACCATGCAGGGTTCCAAGGGAG-3′) and reverse (5′- CTATCCTTCGTTCTCTATTTTGTTG-3′) primers. Amplification conditions were as follows: 98°C (1 min),  × 40 cycles, 72°C (10 min). The amplified fragment was purified using Zymoclean™ Gel DNA Recovery kit . The subsequent sequence was cloned into the vector pENTR (Invitrogen) and the construct was transformed into competent One Shot TOP10 E. coli cells (Invitrogen). The plasmid DNA was isolated from a positive transformant using QIAprep Spin Miniprep Kit (Qiagen) according to the manufacturer’s protocol. The sequence was confirmed  to share 100% identity with rat Slco4c1 sequence (GeneBank accession number NM_001002024). The plasmid DNA was then subcloned into the multiple cloning site of the mammalian expression vector pcDNA6.2/GFP-DEST (Invitrogen).Total RNA was isolated from a frozen kidney of a Sprague-Dawley rat using RNeasy Kit  as directed by the manufacture including on-column DNA digestion, and then reverse-transcribed to cDNA using ThermoScript™ RT-PCR kit (Invitrogen) with oligo (dT)2. LLC-PK1 cells (kind gift from Dr. Alferd Schinkel Madin-Darby canine kidney II (MDCKII) cells  were cultured in minimum essential medium with Earle’s salts  supplemented with 5% fetal bovine serum , 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen) at 37°C in a humidified incubator with 5% COCell lysates were prepared from Oatp4c1-expressing and vector-transfected MDCKII cells. Following a wash with ice-cold PBS, cells were scraped and pelleted by centrifugation at 300 g for 5 min. Cell pellets were resuspended in RIPA buffer  with protease inhibitor cocktail (Roche Diagnostics Co.) and shaken at 4°C for 30 min.2, protease inhibitor cocktail, pH 7.6) with brief pulses of sonication using a probe ultrasonic processor (Thermo Fisher Scientific) at 4°C and tonicity was restored to 150 mM with NaCl. Following 300 g centrifugation for 5 min, EDTA was added to the supernatant to a final concentration of 5 mM. The samples were centrifuged at 100,000 g for 1 hr at 4°C. Pellets were resuspended in buffer . Protein concentrations were measured using a bicinchoninic acid assay . Cell lysates and crude membrane fractions were stored at −80°C until use.Crude membrane fractions were prepared from Sprague-Dawley female rat tissues. The tissues were disrupted in Dounce Buffer  and 0.1 M dithiothreitol before incubation at 70°C for 10 min. Proteins were separated on a 10% SDS-polyacrylamide gel with 5% stacking gel and transferred to a polyvinylidene difluoride membrane  at 200 mA for 2 hr at 4°C. Following blocking with 5% skimmed milk in phosphate buffered saline with 0.05% Tween-20 (PBST) for 3 hr at room temperature, the membranes were incubated with 55 µg/ml anti-Oatp4c1 polycolonal antibody (PA1343) in 5% skimmed milk in PBST overnight at 4°C. After washing with PBST, membranes were incubated with HRP conjugated goat anti-rabbit IgG (Thermo Fisher Scientific) in 5% skimmed milk in PBST for 1 hr at room temperature. Following washing with PBST, a standard ECL kit  and Image Station 2000 MM  were used for visualization.2 growth area, Corning Costar, Lowell, MA) for 4 days to achieve transepithelial electrical resistance >200 Ω•cm2. Filter membranes were washed with ice-cold PBS, excised and processed for staining. After fixation with −20°C methanol for 10 min, rehydration with PBS for 5 min, permeabilization (0.2% triton X-100 in PBS) for 15 min, and blocking with 10% goat serum for 1 hr, cells were incubated with the rabbit anti-Oatp4c1 polyclonal antibody  and mouse anti-ZO-1 monoclonal antibody (5 µg/ml) for 2 hr at room temperature. Following washes with permeabilization solution, cells were incubated with 4 µg/ml Alexa Fluor 568 goat anti-rabbit IgG and Alexa Fluor 488 anti-mouse IgG (Invitrogen) for 1 hr at room temperature. Cells were washed with permeabilization solution and mounted on glass slides with Prolong Gold® containing DAPI solution (Invitrogen) to visualize nuclei. The fluorescence emission was visualized using Axiovert 200 M confocal microscope .Oatp4c1-expressing and vector-transfected MDCKII cells were grown on Snapwell polycarbonate filter membranes  for 4 days to achieve transepithelial electrical resistance >200 Ω•cm2. Cells were washed with ice-cold PBS-Mg/Ca buffer (PBS buffer containing 0.1 mM CaCl2 and 1 mM MgCl2). Freshly prepared Sulfo-NHS-SS-Biotin (1 mg/ml in PBS with Mg++/Ca++) was added to either the apical or basolateral chamber and incubated with slow rocking at 4°C for 20 min. Cells were then washed and incubated with quenching buffer (5 mM glycine in PBS-Mg/Ca) with slow rocking at 4°C for 20 min to remove unreacted biotin. Following washing with PBS with Mg++/Ca++, cells were lysed with 700 µl lysis buffer  for 30 min at 4°C with shaking. Cell lysates were centrifuged at 10,000 g for 2 min at 4°C and supernatants were incubated with streptavidin agarose beads with end-over-end rotation for 1 hr at 4°C. Samples were washed three times with lysis buffer and incubated with 50 µl 2X Laemmli Buffer  for 30 min at room temperature. 2-β-mercaptoethanol was used to break the sulfhydryl (SS) bond between the biotin and the protein reactive group releasing the biotinylated membrane proteins from the beads. Samples were centrifuged at 10,000 g for 2 min, and the supernatants were subjected to western blot analysis.Oatp4c1-expressing and vector-transfected MDCKII cells were grown on Transwell polycarbonate filter membranes  for 15 min each. Non-specific interactions were blocked with 2% normal goat serum in PBST for 30 min prior to incubating with 220 µg/ml anti-Oatp4c1 antibody (PA1343) for 24 hr at room temperature. For visualization, sections were incubated with 5 µg/ml biotinylated goat anti-rabbit IgG (Vector Laboratories) for 30 min at room temperature, Streptavidin-HRP reagent  for 20 min and NovaRED chromogen substrate (Vector Laboratories) for 5 min. After counterstaining with hematoxylin and dehydration, the sections were mounted with Vecta-Mount (Vector Laboratories).Female Sprague-Dawley rat liver and kidney were isolated and fixed in 4% paraformaldehyde for 6 hr at room temperature. The tissues were embedded in paraffin using standard histology techniques, and cut as 6 µm-thick sections. After deparaffinization and rehydration in xylene and ethanol gradient solutions, the antigen was retrieved under pressure in 10 mM citrate buffer, pH 6.0. Sections were blocked with 3% H28k (40 µg/ml), anti-Abcg2 , anti-Mrp4 (3 µg/ml), anti-P-gp (2.5 µg/ml). For visualization, the sections were then incubated with fluorescence conjugated secondary antibodies for 30 min at room temperature and then mounted with Prolong Gold® DAPI containing solution. The fluorescence emission was visualized using Axiovert 200 M confocal microscope .To further study the localization of Oatp4c1, the rat tissue sections were double stained with anti-Oatp4c1 antibody  and the following antibodies: anti-E-cadherin (2 µg/ml), anti-AQP1 (10 µg/ml), and anti-calbindin-D2 was added to yield a final concentration of 12 mM. The mixture was incubated on ice for 15 min with intermittent and gentle mixing. Remaining cellular debris were removed by centrifuging at 3,000 g for 10 min at 4°C, the resultant supernatant was centrifuged at 30,000 g for 40 min at 4°C to isolate membrane proteins. The BBM pellet was resuspended in mannitol buffer and again precipitated by addition of 12 mM MgCl2 as described above. The final pellet was resuspended in mannitol buffer. The BBM and BLM were stored at −80°C until use. Separation efficiency was evaluated by western blot analysis with anti- Na+/K+-ATPase (1∶1000), anti-Mrp4 (3 µg/ml) and anti-Abcg2 (150 µg/m).The BBM and BLM were isolated by Percoll density gradient centrifugation according to procedures described by Goldinger et al. with some modifications 2O, 0.1% formic acid) and mobile B . Eluted peptides were analyzed using data-dependent acquisition: peptide mass spectrometry was obtained by Orbitrap with a resolution of 60,000. The seven most abundant peptides were subjected to collision induced dissociation and MS/MS analysis in LTQ linear trap. The LC-MS/MS data were submitted to a local MASCOT server for MS/MS protein identification search via the ProteomeDiscoverer software. The mass error tolerance was 5 ppm for peptide MS and 0.8 Da for MS/MS. All peptides were required to have an ion score greater than 30 (P<0.05). The false discovery rate in each LC-MS/MS analysis was set to be less than 1%.Approximately 100 µg of proteins from the BBM and BLM were subjected to 10% SDS-PAGE separation. The protein gel bands at ∼65–100 and ∼100–150 kDa were excised and underwent dithiothreitol reduction, iodoacetamide alkylation, and in-gel trypsin digestion using a standard protocol as previously reported 3, 4.83 mM KCl, 0.96 mM KH2PO4, 1.20 mM MgSO4, 5.0 mM glucose and 1.53 mM CaCl2. Buffer pH was adjusted to 7.4 by supplementation with 12.5 mM HEPES and pH adjustment with 1 M Tris-base. Alternatively, the buffer was supplemented with 12.5 mM 2-(N-morpholino)ethanesulfonic acid and pH was adjusted to 4.5 or 5.5 with 1 M Tris-base. Before the study, cells were washed once and preincubated with uptake buffer for 15 min at 37°C. Uptake was initiated by replacing the uptake buffer with that containing [3H]-E3S. After incubation for designated time at 37°C, cells were washed three times with ice-cold PBS, lysed with 0.5 ml of 0.5 N NaOH and neutralized with 0.1 ml of 2.5 N HCl. The intracellular radioactivity was determined by transferring 0.5 ml of lysate into scintillation cocktail  for counting with a Packard 2200 CA Tri-Carb  liquid scintillation counter. Protein concentrations were measured using the BCA Protein Assay kit. Net uptake was calculated as the difference between [3H]-E3S uptake into Oatp4c1-expressing cells and uptake into the vector-transfected cells. For [3H]-E3S uptake under ATP-depletion conditions, cells were pre-incubated for 20 min prior to the experiment and during the experiment (1 min) with uptake buffer containing 20 mM 2-deoxy-D-glucose and 10 mM NaN3 without D-glucose 3H]-E3S uptake inhibition, cells were pre-incubated for 15 min prior to and during the experiment (1 min) with 100 µM of various compounds at pH 5.5 or 7.4. Based on model fitting criteria, transport parameters were estimated using a sigmoid-Emax equation:max, S50, and n represent the initial uptake velocity, substrate concentration, maximum uptake transport velocity, Michaelis-Menten constant equivalent, and Hill coefficient, respectively. The model was fitted to the data (obtained in triplicate) by nonlinear regression using GraphPad Prism .Oatp4c1-expressing and vector-transfected MDCKII cells were grown in 6-well plates. The cell culture medium was replaced with that containing 5 mM sodium butyrate to induce transporter expression 24 hr prior the uptake study Values are presented as means ± S.D. Statistical analysis was performed with unpaired student’s t-test or two-way ANOVA and Bonferroni post-hoc test for multiple comparisons. p<0.05 was the criterion of statistical significance. GraphPad Prism was used for all statistical analysis .To study the role of rat Oatp4c1 in drug disposition, we generated rat Oatp4c1-expressing MDCKII cells. The expression of Oatp4c1 in the transfected cells were verified by western blotting using the antibody (PA1343) generated against C-terminal of rat Oatp4c1 . Oatp4c1+/K+-ATPase demonstrated the efficient separation of the apical and basolateral membranes. Western blot analysis of Oatp4c1 demonstrated that the transporter was enriched in the apical membrane fraction, but not in the basolateral membrane , a basolateral marker, was detectable in both membrane fractions, but more so in BLM than BBM. Oatp4c1 was only detected in BBM  in each membrane preparation are also shown. Eleven apical proteins were predominantly in the BBM, four basolateral proteins were enriched in the BLM, and three proteins with previously reported apical localization were found in both fractions (ambiguous). Four unique Oatp4c1 peptides  were found only in the BBM. These qualitative data provide additional, but conclusive evidence that Oatp4c1 localizes at the apical membrane in the rat kidney cortex.To validate the specificity of the PA1343 antibody, the proteome in BBM and BLM were analyzed by LC-MS/MS. The proteins in BBM and BLM were separated by SDS-PAGE. The gel bands of proteins eluting at 65–100 and 100–150 kDa were excised, and underwent reduction, alkylation, and in-gel trypsin digestion 28K28K. Therefore, Oatp4c1 is expressed in the proximal tubule within the juxtamedullary cortex, which suggests that it localizes primarily in the proximal straight tubules (S3). Double immunofluorescence staining was also used to access the location of Oatp4c1 relative to major apical efflux transporters in the rat kidney. As shown in To further gain insights on the potential physiological role of Oatp4c1 we assessed its precise expression pattern at the organ and nephron level. Immunohistochemical analysis showed that Oatp4c1 is expressed densely in the renal tubules located in the juxtamedullary cortex . To asce3H]-E3S in MDCKII-Oapt4c1 cells was susceptible to inhibition by 100 µM unlabeled E3S and transport appeared to be more efficient in cells exposed to media with pH 5.5 , under pathological situations. Our studies showed that the time dependent uptake of [3H]-E3S (0.5 µM) was more efficient in Oatp4c1 cells as compared to vector transfected cells at pH 5.5  was comparable to the reported Km (26.6±4.9 µM) for human OATP4C1 at pH 7.4 50 with a decrease in pH while the estimated Tmax was 5- and 4-fold higher at pH 4.5 and pH 5.5, respectively, than the Tmax estimate at pH 7.4. In addition, the estimated Hill coefficient value of 2.6 at pH 7.4 suggests positive transport cooperativity.To obtain quantitative estimates of the pH dependent differences in transport, we then examined the concentration dependent uptake (1 min) of -E3S concentration. As shown in 50 value, but decreases rapidly as the substrate concentration saturates the transporter. This analysis also demonstrates that the transporter uptake clearance is 10–20 fold higher at acidic pH conditions.Using the estimated transport parameters we then determined the uptake transport clearance as a function of [3H]-E3S (0.5 µM) uptake was determined in the absence and presence of 100 µM various compounds for 1 min at pH 5.5  in both Oatp4c1-expressing and vector-transfected cells  was present ondition . In addit pH 5.5 , suggestIn the present study, we undertook a comprehensive approach to determine the subcellular localization of Oatp4c1 in polarized epithelium. Our studies definitively demonstrated that Oatp4c1 is expressed at the apical membrane in the rat proximal tubule. Our approach included a variety of complementary methods to verify this observation. Western blotting demonstrated that the antibody used recognizes a protein of the appropriate molecular weight. Immunohistochemical and immunofluorescence analysis revealed that the protein traffics to the apical membrane of polarized epithelium in vivo and in vitro. The immunohistochemistry results were verified with four different antibodies, which were raised against unique C- and N-terminal peptides. In addition, biochemical separation of apical and basolateral membranes from polarized cells in vitro and proximal rat kidney tubules confirmed the location of Oatp4c1 at the brush border membranes by western blot and proteomic analyses. Furthermore, the proteomic analyses provide additional validation for the Oatp4c1 antibody specificity.This extensive validation of the in vitro trafficking and in vivo subcellular localization was deemed necessary as our results were not in accordance with previous reports. Initially, Oatp4c1 was shown to localize at the basolateral membrane in the rat proximal tubule +/K+-ATPase or, rBat, SGLT2, TSC or NKCC2, were detectable in both membrane fractions, showing lack of absolute efficiency in the separation of the two membrane fractions. Cross-contamination between the two fractions was also observed in previous reports using the same method It is not without precedent that antibodies used to detect transporter proteins result in controversial findings Previous studies have shown the expression of several organic anion transporters in the kidney 4 and enhanced by T3, suggesting that T4 is a substrate/inhibitor and T3 is an enhancer. Both T3 and T4 have been reported as OATP4C1 substrates 3 decreases OATP4C1-mediated E3S transport at pH 7.4 3 and digoxin 3, and another that recognizes digoxin. The identification of Oatp4c1 substrates should be investigated further so that its physiological role as well as its function in the renal disposition of drugs and/or their metabolites can be understood.In this study, we found Oatp4c1 is expressed primarily at the apical membrane in the proximal straight tubule (S3), suggesting its potential role in renal reabsorption of substrates. In agreement with previous studies, we showed that E3S is an Oatp4c1 substrate. Oatp4c1-mediated E3S uptake was inhibited by TWith respect to physiological substrates, Toyohara and colleagues showed that OATP4C1 transgenic rats had decreased uremic toxin plasma concentrations in the 5/6 nephrectomy renal failure model as compared to the same model established wild-type rats 3 uptake are significantly higher at extracellular pH 6.5 than pH 8.0 in Xenopus laevis oocytes expressing OATP4C1 max was 4-fold higher at pH 5.5 than at pH 7.4 and uptake clearance was 10–20 fold higher at acidic pH. The observed pH-dependent Oatp4c1-mediated uptake may be a consequence of altered ionization state of the substrate molecule. However, with a pKa of 2 3− exchange. Further studies examining substrate uptake in HCO3− replete and depleted conditions are needed to confirm the potential role of an organic anion/HCO3− exchange mechanism. In addition, the pH in renal proximal tubules of rats is acidic The pH dependence of OATP mediated transport has been demonstrated in several rodent and human OATP proteins in vitro cell models , as well as in the rat kidney proximal tubules, which suggests that Oatp4c1 is involved in renal reabsorption. In conclusion, our data collectively demonstrate that rat Oatp4c1 polarizes to the apical membranes in Figure S1Immunolocalization of Oatp4c1 in polarized LLC-PK1 cells. Cells were double stained with Oatp4c1 (red) and ZO-1 (green). Nuclei were stained with DAPI (blue). Center image in the Oatp4c1 panel is a single optical section of the x–y plane while top and right images represent x–z and y–z planes, respectively, reconstructed from image stacks. The apical and basal sides can be demarcated by ZO-1 and the nuclei, respectively, in both x–z and y–z sections.(TIFF)Click here for additional data file.Figure S2Effect of sodium butyrate (NaB) on Oatp4c1 expression and subcellular localization in MDCKII cells. MDCKII-pcDNA and MDCKII-Oatp4c1 cells were treated with 1 or 5 mM NaB for 24 hr. Oatp4c1 expression (red) was assessed in cells grown in monolayers (A) and cell pellets were collected, lysed and subjected to PCR for Slco4c1 mRNA expression (B) or western blot for Oatp4c1 protein expression (C). 18 s and β-actin were used as loading controls in panels B and C, respectively. Oatp4c1 subcellular localization was assessed in polarized MDCKII cells by confocal microscopy (D). After treatment with 5 mM NaB for 24 hr, cells were double stained with Oatp4c1 (red) and ZO-1 (green). Nuclei were stained with DAPI (blue). Center image in the Oatp4c1 panel is a single optical section of the x–y plane while top and right images represent x–z and y–z planes, respectively, reconstructed from image stacks. The apical and basal sides can be demarcated by ZO-1 and the nuclei, respectively, in both x–z and y–z sections.(TIFF)Click here for additional data file.Figure S3Apical Oatp4c1 localization in rat kidney tubules was verified by four different antibodies. Paraformaldehyde-fixed paraffin-embedded rat kidney tissue sections were stained with different rabbit polyclonal anti-Oatp4c1 antibodies, as indicated. Color development with NovaRed signifies Oatp4c1 staining. All sections were counterstained with hematoxylin. Rabbit IgG was used as a negative control. Antibody specificity (PA1343) was also demonstrated by pre-absorbing the antibody with antigen peptide (STITVEEDLNKIENEG) overnight at 4°C prior to use. PA1556 was generated against the peptide (SPDFEARAGKC) previously reported by Mikkaichi and colleagues (TIFF)Click here for additional data file.Figure S43H]-E3S is inhibited by E3S.Oatp4c1 mediated uptake of [ MDCKII-pcDNA and MDCKII-Oatp4c1 cells were incubated with 0.5 µM [3 H]-E3S in the absence (control) and presence of 100 µM unlabeled E3S for 1 min at pH 5.5 (black bars) and 7.4 (white bars). Oatp4c1 mediated uptake was calculated after subtraction of nonspecific uptake by pcDNA cells. Each column represents the mean ± S.D. of triplicates. Statistical analysis was performed with unpaired student’s t-test. *p<0.05, significant differences from control.(TIFF)Click here for additional data file.Figure S53H]-E3S uptake by various compounds.Inhibition of [ MDCKII-pcDNA and MDCKII-Oatp4c1 cells were incubated with 0.5 µM [3H]-E3S in the absence (control) and presence of various compounds (100 µM) for 1 min at pH 5.5 (A) and 7.4 (B). Each point represents the mean ± S.D. of triplicates.(TIFF)Click here for additional data file.Figure S63H]-E3S uptake via Oatp4c1.Effect of ATP on [ (A) MDCKII-pcDNA (white bars) and MDCKII-Oatp4c1 cells (black bars) were incubated with 0.5 µM [3H]-E3S for 1 min at pH 5.5 and pH 7.4. Twenty minutes prior to the transport experiment, and for the duration of transport, cell medium was replaced with medium that contained 20 mM 2-deoxy-D-glucose and 10 mM NaN3 without D-glucose. (B) Oatp4c1-mediated uptake was calculated after subtraction of nonspecific uptake by pcDNA cells. Each column represents the mean ± S.D. of triplicates.(TIFF)Click here for additional data file.


Hepatitis C virus (HCV) subgenotypes 1a and 1b have different impacts on the treatment response to peginterferon plus ribavirin with direct-acting antivirals (DAAs) against patients infected with HCV genotype 1, as the emergence rates of resistance mutations are different between these two subgenotypes. In Japan, almost all of HCV genotype 1 belongs to subgenotype 1b.To determine HCV subgenotype 1a or 1b in Japanese patients infected with HCV genotype 1, real-time PCR-based method and Sanger method were used for the HCV NS5B region. HCV subgenotypes were determined in 90% by real-time PCR-based method. We also analyzed the specific probe regions for HCV subgenotypes 1a and 1b using ultra-deep sequencing, and uncovered mutations that could not be revealed using direct-sequencing by Sanger method. We estimated the prevalence of HCV subgenotype 1a as 1.2-2.5% of HCV genotype 1 patients in Japan.Although real-time PCR-based HCV subgenotyping method seems fair for differentiating HCV subgenotypes 1a and 1b, it may not be sufficient for clinical practice. Ultra-deep sequencing is useful for revealing the resistant strain(s) of HCV before DAA treatment as well as mixed infection with different genotypes or subgenotypes of HCV. Flaviviridae family. HCV genomes are translated into a single open reading frame of ~3000 amino acids, and by cellular and viral encoded-protease are processed into structural  and non-structural proteins   and four mutations  were identified in ultra-deep sequences. Similarly, in No. 78, one mutation [8922.5:/G (1.09%)] was identified .+ 0.14 vs. 0.074 + 0.029, P = 0.11).Even in the HCV subgenotype 1a isolate, analysis of ultra-deep sequences showed one nucleotide sequence variation (1%), compared to MGB probe sequence, according to HCV H77 . We alsoIn the present study, we used a novel HCV subgenotyping method based on real-time PCR  in JapanUltra-deep pyrosequencing is a promising technology for characterizing and detecting minor variants and drug resistant variants . PyroseqWe estimated the prevalence of HCV subgenotype 1a at 1.2-2.5% of HCV genotype 1 patients in Japan. It is well known that some patients treated with telaprevir experienced viral breakthrough with telaprevir-resistant strains, most of which harbored the R155T/K mutations. Only a single nucleotide change is needed to produce the amino acid change at codon 155 in HCV subgenotype 1a, while two nucleotide changes are required in HCV subgenotype 1b. This might be one of the reasons why resistance mutation was selected more frequently in HCV subgenotype 1a than in HCV subgenotype 1b in the case of telaprevir use , althougIt was reported that Versant HCV genotype assay  could not determine HCV subgenotypes in 23% of HCV-infected patients in Brazil . Thomas It was reported that phylogenetic analysis revealed different clusters between the HCV subgenotype 1b strains obtained in Japan and those in Brazil . In factIn conclusion, the method for identifying HCV subgenotype 1a or 1b by the real-time PCR-based method in the HCV NS5B region was examined in Japanese patients infected with HCV genotype 1, and we estimated a 1.2-2.5% prevalence of HCV subgenotype 1a among HCV genotype 1 patients in Japan. We also analyzed the specific probe regions for HCV subgenotypes 1a and 1b, of which we could not determine HCV subgenotypes using specific probes, and uncovered the mutations that direct-sequencing by the Sanger method could not reveal, existing in their probe regions using ultra-deep sequencing. Ultra-deep sequencing is useful for revealing the resistant strain(s) of HCV before DAA treatment as well as mixed infection with different genotypes or subgenotypes of HCV.Sera from 80 consecutive treatment-naïve chronic hepatitis C genotype 1 patients with well-characterized clinical follow-up were selected from a chronic hepatitis C database at Chiba University Hospital, Chiba, Japan. All patients were negative for hepatitis B and HIV antibodies. Sera from patients were stored at -20°C until analysis. This study was approved by the Ethics Committee, Chiba University, Graduate School of Medicine, Chiba, Japan (permission numbers 1462 and 282), and conformed to the Helsinki Declaration. Written informed consent was obtained from all patients before enrollment in this study.Total RNA was extracted from 140 µL of each serum sample using a QIAamp Viral RNA Mini Kit  according to the manufacturer’s instructions. RNA was eluted in 60 µL of elution buffer and quantified using a NanoDrop Lite spectrophotometer .4, and 0.4 mM of each dNTP), 1.0 µL of Taq mix , 200 nM of reverse primer (R56_1), and 0.5 µL of RNase Out (Invitrogen). Twenty-one microliters of F1 was added to optical tubes. The second reaction mix fraction (F2) consisted of 4.4 µL of 2X reaction mix, 200 nM of forward primer (F56_1), 1.5 µL of 50 mM MgSO4, 0.1 µL of ROX (25 µM), and 200 nM of each probe (against either genotype 1a or 1b). A final F2 volume of 10 µL was transferred to another optical tube. After the addition of 19 µL of RNA, F1 was carefully added to F2. Reverse transcription was carried out in a TakaRa PCR Thermal Cycler  at 55°C for 35 min with the thermocycler cover open. Following this, the tubes were briefly centrifuged and real-time PCR was carried out in a 7300 Real-Time PCR System  using the following cycling parameters: 50°C for 2 min; 95°C for 10 min; and 40 cycles at 95°C for 15 sec, 50°C for 30 sec, and 60°C for 1 min. The total time required for performing this assay was only 2 hours, as previously described [Reactions were performed in a final volume of 50 µL using the Superscript™ III Platinum one-step quantitative RT-PCR system  as previously described . The desescribed .To perform ultra-deep sequencing of the MGB probe region in HCV NS5B, we used the HPLC-purified specific primers shown in 7 molecules of these final DNA libraries were then subjected to emulsion PCR, and enriched DNA beads were loaded onto a picotiter plate and pyrosequenced with a Roche/454 GS Junior sequencer using Titanium chemistry . GS Amplicon Variant Analizer Version 2.7 (Roche) was used for read mapping and calculating variant frequencies at each nucleotide position according to reference sequence HCV subgenotype 1a, strain H77 or HCV subgenotype 1b, strain Con1.Then, the first PCR product was further amplified with two inner primer sets using the FastStart high-fidelity PCR system, dNTPack kit (Roche). Set one was Pr2 (sense primer) and R56_1 (antisense primer), and the other was Pr1 (sense primer) and Pr2 (antisense primer). Considering the MGB probe locations at nt 8913-8926(1a) in reference sequence HCV subgenotype 1a, strain H77 (AF009606.1), and nt8910-8923(1b) in reference sequence HCV subgenotype 1b, strain Con1 (AJ238799.1), primer set one was used for PCR. The PCR conditions were as follows: 35 cycles at 95°C for 30 sec, 55°C for 30 sec, and 72°C for 60 sec. Amplified products were separated by agarose gel electrophoresis and purified using a high pure PCR clean-up micro kit (Roche). Each amplicon was quantified using a NanoDrop Lite spectrophotometer (Thermo Scientific), and all amplicons from a single viral genome were pooled together at equimolar ratios. Each pool was then quantitated, and approximately 500 ng of each was used in a fragmentation reaction mix using a GS FLX Titanium Rapid Library Preparation Kit (Roche). Final libraries representing each genome were characterized for average size by using an Agilent High Sensitivity DNA kit on Agilent 2100 Bioanalyzer . 4 x 10Then, we amplified the first PCR product using primer set one and TaKaRa Ex Taq (TaKaRa). The PCR conditions were as follows: 40 cycles at 98°C for 10 sec, 55°C for 30 sec, and 72°C for 60 sec; the last cycle followed at 72°C for 7 min. Sanger sequencing was performed using a BigDye(R) Terminator v3.1 Cycle Sequencing Kit . Sequences were detected using Applied Biosystems 3730xl. Nucleotide sequences were analyzed by GENETYX 10  and imported to MEGA version 4 , which wA fragment of about 424-bp length containing the HCV NS5B region was used for phylogenetic analysis. Phylogenetic trees were inferred using the Neighbor-Joining (NJ) model and robustness of the tree branches was tested using bootstrap analysis (500 replicates) with MEGA version 4 .DNA Data Bank of Japan (DDBJ) Sequence Read Archive under accession number DRA001077.All sequence reads have been deposited in the + standard deviation. We used univariative analyses, applying Student’s t-test or Chi-square test as appropriate. P 0.05 was considered statistically significant.Data were expressed as mean


Precision-cut lung slices of pigs were infected with five swine influenza A viruses of different subtypes . The viruses were able to infect ciliated and mucus-producing cells. The infection of well-differentiated respiratory epithelial cells by swine influenza A viruses was analyzed with respect to the kinetics of virus release into the supernatant. The highest titres were determined for H3N2/2006 and H3N2/2007 viruses. H1N1/1981 and H1N2/2000 viruses replicated somewhat slower than the H3N2 viruses whereas a H1N1 strain from 2006 multiplied at significantly lower titres than the other strains. Regarding their ability to induce a ciliostatic effect, the two H3N2 strains were found to be most virulent. H1N1/1981 and H1N2/2000 were somewhat less virulent with respect to their effect on ciliary activity. The lowest ciliostatic effect was observed with H1N1/2006. In order to investigate whether this finding is associated with a corresponding virulence in the host, pigs were infected experimentally with H3N2/2006, H1N2/2000, H1N1/1981 and H1N1/2006 viruses. The H1N1/2006 virus was significantly less virulent than the other viruses in pigs which was in agreement with the results obtained by the in vitro-studies. These findings offer the possibility to develop an ex vivo-system that is able to assess virulence of swine influenza A viruses. Influenza A viruses are a major cause of acute respiratory disease in pigs. Typical disease is characterized by high fever, loss of appetite, depression, tachypnoea, abdominal breathing and, less frequently, coughing. Mortality rates are low, but morbidity rates can be as high as 100% .Swine influenza virus (swFLUAV) strains that are endemic in swine populations worldwide are assigned to the subtypes H1N1, H3N2, or H1N2. Depending on the geographic distribution, these viruses differ in their origin as well as in their genetic and antigenic properties . This swPrimary target cells for influenza viruses are cells of the respiratory epithelium . In vitr2 at 37 °C and passaged every 2–3 days.MDKC II, a subline of Madin-Darby canine kidney cells  were maig, 10 min) and stored at -80 °C.Swine influenza viruses of the H1N1 subtype (A/sw/Potsdam/15/1981) H1N1/1981, the H1N2 subtype (A/sw/Bakum/1832/2000) H1N2/2000 and the H3N2 subtype (A/sw/Herford/IDT5932/2007) H3N2/2007 were provided by Prof. Michaela Schmidtke, University of Jena, Germany. The H1N1 subtype (A/sw/Bad Griesbach/IDT5604/2006) H1N1/2006 and the H3N2 subtype (A/sw/Damme/IDT5673/2006) H3N2/2006 were obtained from IDT Biologika GmbH, Dessau-Rosslau, Germany. The strains had been originally isolated from pigs with respiratory disease during surveillance programs initiated by Prof. Jochen Süss, Berlin (strains Potsdam and Bakum) and Dr. Ralf Dürrwald, Dessau-Rosslau . Virus stocks were propagated by infection of MDCK cells at low multiplicity of infection and incubation in infection medium ) containing acetylated trypsin 1 μg/mL . Supernatants were clarified by low-speed centrifugation  as previously described ,25,27. A-4 M methacholine  as described previously [The viability was analyzed by observing the ciliary activity under the light microscope (Zeiss Axiovert 35) equipped with an ORCA C4742-80 digital camera (Hamamatsu) and SIMPLE-PCI analysis software (Compix Imaging Systems). In selected samples, the slices were analyzed for bronchoconstriction by addition of 10eviously ,25. The 5 TCID50/mL (50% tissue culture infectious dose/mL). Supernatants from virus-infected and uninfected control slices were collected at different time points  post-infection and the samples were stored at -80 °C. For virus infectivity analysis, virus titers were determined by endpoint dilution titration on MDCK cells in 96-well plates as described previously [For infection experiments, three slices were used for each virus and all trials were repeated three times. PCLS were infected by different subtypes of swine influenza viruses  in a volume of 300 μL/slice at 10eviously . BrieflyPCLS were analyzed under a light microscope to estimate the ciliary activity as described previously . Each brPCLS were mounted on small filter paper with tissue-freezing medium , frozen in liquid nitrogen and kept in -80 °C prior to cutting. Slices were cut at 10 μm thickness by a cryostat . The sections were dried overnight at room temperature and kept frozen at -20 °C until staining.Sections with a central bronchus were fixed with 3% paraformaldehyde for 20 min followed by 5 min incubation with 0.1 M glycine and three washing steps with PBS. Then, the sections were permeabilized with 0.2% Triton X-100 for 20 min at room temperature followed by 3 washing steps with PBS. All antibodies were diluted in 1% bovine serum albumin and incubated with the sections for 1 h at room temperature in a humid incubation chamber. For detection of virus particles, monoclonal antibody against the influenza A virus nucleoprotein (NP)  at a 1:750 dilution was used followed by incubation with anti-mouse IgG (Sigma-Aldrich) secondary antibody. For detection of infected cells, ciliated and mucus-producing cells were stained by antibodies against β-tubulin and mucin, respectively ,20,23,247 TCID50/m3).All trial procedures and animal care activities were conducted under approval of Good Clinical Practice  and German Animal Protection Law at IDT Biologika GmbH. The protocol IDT A 3/2004 was approved by the Landesverwaltungsamt Sachsen-Anhalt (Reference Number: AZ 42502-3-401 IDT). A total of 48 crossbred swine  were used in the present study. Pigs had been proved to be free of influenza during their life span as well as free of maternally-derived antibodies against influenza A viruses. They were housed in identical isolation rooms based on their challenge status and were provided with feed and water ad libitum. Experimental infections were done in the BSL-2 infection units of IDT Biologika GmbH, Dessau-Roβlau, Germany, which are equipped with High Efficiency Particulate Airfilter H13 filters. Four independent trials were performed for each of the four viruses examined. The experimental design is summarized in Table After infection, rectal temperatures and signs of respiratory disease, dyspnoea, and cough were recorded twice daily 1–3 days post-infection (dpi). Dyspnoea was assessed as follows: 1, increased respiratory frequency and moderate flank movement; 2, marked breathing difficulty and severe flank movement; 3, laboured breathing affecting the entire body, pronounced flank movement and substantial movements of the snout; 4, extreme breathing difficulty reflecting substantial lack of oxygen.10) from lung samples were injected into the allantois cavity of 11-day-old chicken embryos . After sealing the perforation point eggs were incubated at 37 °C and checked daily for vitality using an egg candler. On day 4 post-infection (pi), the allantois fluid was collected and tested in the haemagglutination test. The Spearman and Kaerber method was used to calculate the EID50 from the haemagglutinating activity.In each trial 5–7 animals were stunned by electrical stunning tongs 1 dpi and bled to death. Lung tissue samples were taken from each lobe for virus detection. Samples of the right and left halves of the lungs were pooled, ground with sterile sea sand, and diluted 1:10 in dilution medium . The virus load in the lungs was determined by titration of the lung samples in embryonated hens eggs. Dilution series   and foun6 and 1 × 107 TCID50/mL. These values increased or decreased only slightly by 48 hpi  was not performed because both H3N2 viruses provided similar results in the in vitro investigations. Coughing was only observed rarely and inconsistently and therefore not analyzed.p < 0.001) than those determined for the other three viruses. Strains H1N2/2000, and H3N2/2006 were intermediate; while their dyspnoea scores on day 1 pi were similar to those of H1N1/1981, these values dropped significantly faster on the following two days than those of H1N1/1981 (p < 0.05).With respect to the dyspnoea score, the H1N1/1981 strain was most virulent showing high values even three days after infection Figure . Only lop < 0.001) and to strain H1N2/2000 on day 3 pi (p < 0.05), respectively.Infection of pigs with strain H1N1/2006 did not affect the rectal temperature Figure . Increasp < 0.05).The viral load was determined for both, the right and the left half of the lung Figure . In geneOur analysis of the virulence of swFLUAVs shows that the three strains H3N2/2006, H1N1/1981, H1N2/2000 cause disease in pigs as evidenced by a rise in the parameters dyspnoea, rectal temperature, and virus load. Low virulence was determined for strain H1N1/2006 which showed humble dyspnoea values and no increase in rectal temperature. The analysis of the other three strains shows that disease symptoms may vary as indicated by the finding that changes in one parameter were most prominent for one strain, whereas another strain was affected more at a different parameter. The dyspnoea values decreased in the order H1N1/1981 > H1N2/2000 / H3N2/2006 > H1N1/2006. The rectal temperature decreased in the order H3N2/2006 > H1N2/2000 > H1N1/1981 > H1N1/2006. In the viral lung load, the order was H3N2/2006 / H1N1/1981 > H1N2/2000 > H1N1/2006. Despite these differences there was one remarkable common feature with respect to all parameters: the low virulence of H1N1/2006. Interestingly, strain H1N1/2006 was isolated from a diseased animal. Similar to other current European H1N1 swFLUAV strains, it is characterized by a low virulence in animal experiments . By contPCLS are a promising culture system to analyze the infection of differentiated respiratory epithelial cells. From our data we suggest that the characteristics of infection determined here for four swFLUAV strains reflect the pathogenic properties of these viruses. The strain with a low pathogenicity, A/Bad Griesbach/IDT5604/2006 (H1N1), differed from the other strains not only in a significantly lower amount of virus released into the supernatant but also in a lower ciliostatic effect. It will be interesting in the future to find out whether complete ciliostasis is an indicator for the virulence of swFLUAV, whereas low virulent swFLUAVs may be able only to cause partial ciliostasis. Such a marker would greatly facilitate the screening of treatments or antivirus substances ,33 for tPCLS may also help to distinguish between parameters of virulence. The H3N2 strains were superior to H1N1 and H1N2 strains in both virus release and ciliostatic effect. Among virulence parameters in infected pigs, the H3N2 virus showed the largest effect on rectal temperature and virus load and reflected also a high dyspnoea score. Though more experiments are required to confirm these suggestions, the value of an in vitro-system that predicts virulence is worth the effort.The authors declare that they have no competing interests.FM, DP, IHP, CSW, XR, and GH conceived and designed the experiments; FM, DP, and SU performed the experiments; FM, IHP, CSW, XR, and GH analyzed the data; RD designed, performed and analyzed the infection trials in pigs; IHP and RD contributed reagents/materials/analysis tools; FM, GH, RD and XR wrote or helped to draft the paper. All authors read and approved the final manuscript.