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Bronchiolitis obliterans syndrome is associated with increased p-glycoprotein expression and loss of glucocorticoid receptor from steroid resistant pro-inflammatory CD8+T cells.

Immunosuppressive therapy fails to suppress the production of pro-inflammatory cytokines, particularly by CD8+T cells, in stable lung transplant recipients and those undergoing chronic rejection, suggesting that some patients may become relatively resistant to immunosuppressants such as glucocorticoids (GC). We have shown loss of GC receptor (GCR) from the CD8+ cells, and we hypothesized that the drug membrane efflux pump, p-glycoprotein-1 (Pgp), may also be involved in lymphocyte steroid resistance following lung transplant. Pgp/GCR expression and IFNγ/TNFα pro-inflammatory cytokine production was measured in blood lymphocytes from 15 stable lung transplant patients, 10 patients with bronchiolitis obliterans syndrome (BOS) and 10 healthy aged-matched controls (± prednisolone ± Pgp inhibitor, cyclosporin A ± GCR activator, Compound A) using flow cytometry. Both Pgp+ and Pgp- lymphocyte subsets from all subjects produced IFNγ/TNFα pro-inflammatory cytokines. Pgp expression was increased in CD8+Pgp+T cells and correlated with IFNγ/TNFα expression and BOS grade. Reduced GCR was observed in CD8+Pgp-T, NKT-like and NK cells from stable patients compared with controls, and further reduced in CD8+Pgp-T cells in BOS. Addition of 2.5ng/ml cyclosporine A and 1µM prednisolone significantly inhibit IFNγ/TNFα production by CD8+Pgp+ T cells from BOS patients. Addition of 10µM Compound A and 1µM prednisolone significantly inhibit IFNγ/TNFα production by CD8+Pgp- T cells from BOS patients. BOS is associated with increased Pgp expression and loss of GCR from steroid resistant pro-inflammatory CD8+T cells. Treatments that inhibit Pgp and upregulate GCR in CD8+T cells may improve graft survival. This article is protected by copyright. All rights reserved.

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U-box ubiquitin ligase PPIL2 suppresses breast cancer invasion and metastasis by altering cell morphology and promoting SNAI1 ubiquitination and degradation.

Metastasis is the leading cause of breast cancer fatalities. To develop new therapeutic strategies, the mechanisms underlying breast cancer invasion and metastasis need to be further investigated. Peptidylprolyl isomerase (cyclophilin)-like 2 (PPIL2) is a U-box-type E3 ubiquitin ligase belonging to the cyclophilin family. Proteins within this family are the major cytosolic binding proteins of the immunosuppressant drug cyclosporine A (CsA). Although PPIL2 has been reported to potentially be involved in cell migration, its role in breast cancer is still unclear. Herein, we demonstrate that PPIL2 suppressed metastasis in a breast cancer model by altering cell morphology and suppressing the epithelial-mesenchymal transition (EMT) process. Moreover, elevated PPIL2 inhibited EMT and breast cancer invasion by interacting with the classical EMT transcription factor, SNAI1, to enhance its ubiquitin-dependent degradation. Furthermore, PPIL2 protein level and stability was upregulated after CsA treatment, indicating that PPIL2 might be involved in CsA-mediated repression of EMT in breast cancer. Analysis of tissue samples taken from breast cancer patients showed a significant correlation between the expression of PPIL2 and the degree of cancer invasion and metastasis. In summary, these results would shed light on a potential clinical use of CsA in breast cancer patients.

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Use of rifabutin to treat tuberculosis in a cardiac transplant recipient: A case report
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Tuberculosis is an important concern following organ transplantation. Unfortunately, several antituberculosis drugs interact with immunosuppressants. This report describes our experience with rifabutin (RBT) in the treatment of acute tuberculosis in a cardiac transplant recipient.

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Biomarkers of treatment efficacy in patients with chronic spontaneous urticaria.

Background. Currently there are no biomarkers useful to predict the future evolution and the therapeutic response in patients with chronic spontaneous urticaria (CSU). Objective. To review the available information on biomarkers that might be applied for the follow up of the response to guideline recommended therapies for CSU. Methods. A review of the medical literature on CSU potential clinical and laboratory biomarkers in PubMed and MEDLINE including the terms urticaria, chronic urticaria, chronic idiopathic urticaria, chronic spontaneous urticaria, antihistamines (AHs), omalizumab (OMA), cyclosporine (CyA), and treatment. Results. Clinical manifestations that were associated to poor responses to AHs were atopy, asthma, rhinitis / rhinosinusitis, thyroid disease, hypertension, higher disease activity and duration. Laboratory markers of AH resistance that have been reported include Complement C5a fraction, Autologous Serum Skin Test (ASST), Basophil Activation Test (BAT), D-dimer and LCN2 adipokine. Basophil Histamine Release Assay (BHRA), ASST, and basophil CD203c-upregulating activity in the serum correlated with favorable response to OMA, whereas disease duration and severity, BAT, BHRA, and D-dimer levels were associated with better responses to CyA. Conclusion. Some promising biomarkers useful for patient management in CSU, have been identified in the literature. There is, however, an urgent need of new, easy-to-perform markers that can be made widely available for the optimal care of patients suffering CSU.

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Current and Emerging Therapy on Lupus Nephritis.

Lupus nephritis (LN) is involvement of the kidney in patient with systemic lupus erythematosus (SLE) and one of the most common target organ in SLE. The diagnosis of LN will significantly impact the clinical outcome and therapy of the patient. Therapy regiment of LN is divided into two stages, induction and maintenance treatment. The main objective of the induction therapy is to achieve complete or partial remission as soon as possible since it is correlated with better prognosis and fewer relapse incidence. In the maintenance stage, the main aim of the therapy is to maintain the remission status and avoid future relapse. It is also important to evaluate the effectiveness of the therapy as it will affect the duration and the regiment therapy being used. Corticosteroid, cyclophosphamide, mycophenolate mofetil, azathrioprine, cyclosporine and tacrolimus are example of drugs used in LN therapy. Currently, studies are being conducted to evaluate and develop targeted drug therapy to further add treatment options for LN.

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Recurrent IgA NEPHROPATHY after renal transplantation AND STEROID WITHDRAWAL.

Immunoglobulin A Nephropathy (IgAN), is the most common primary glomerulonephritis; the reported recurrence rate of IgAN after renal transplantation is as high as 13-50%. The impact of immunosuppressive therapy and steroid withdrawal on the risk of recurrence of IgAN is still under debate. We performed a retrospective single center study, selecting 123 kidney transplants (rtx) in 120 patients, between January 1995 and December 2012, with IgAN on the native kidney. In 51/123 at least one post transplantation biopsy for clinical indication was performed; in 28/51 transplants IgAN recurrence (IgANr) was demonstrated. This group (G1; N = 28) was compared with a group without IgANr (G2; N = 23). In our study, clinically evident IgANr rate was 54.9% (28/51) on biopsied patients. At discharge the use of the immunosuppressant drugs (tacrolimus, cyclosporine A, mycophenolate mofetil, azathioprine, mTor inhibitors) was not associated with an increased risk of IgANr (P=NS). At discharge, all patients were steroids treated. Neither the use of tacrolimus, mycophenolate mofetil or mTor inhibitors (mTori) at biopsy time were associated with IgANr. However, IgANr was significantly higher in patients who experienced steroid withdrawal at any post transplantation time (OR 7,7 p =0,03). The median time to recurrence after steroid withdrawal was 59 months (min 4.18, max 113.2). This article is protected by copyright. All rights reserved.

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Growth of human breast cancers in Peromyscus.

Modeling breast cancer in general and hormone-sensitive breast cancer, in particular in mice, has several limitations. These are related to the inbred nature of laboratory mice, and do not allow adequate appreciation of the contribution of the host's genetic heterogeneity in tumor growth. In addition, the naturally low estrogen levels of mice makes estradiol supplementation obligatory for tumor growth. Here, we show that Peromyscus californicus, following cyclosporine-mediated immunosuppression, supports the growth of both MDA-MB-231 estrogen-independent and MCF7 estrogen receptor-positive breast cancers without exogenous estradiol supplementation. Tumor growth was inhibited by fulvestrant or letrozole, confirming that MCF7 xenografts remain hormone dependent in vivo and suggesting that P. californicus can be used as an alternative to conventional mice for the study of hormone-sensitive breast cancer. The fact that Peromyscus stocks are outbred also facilitates the study of breast cancer in genetically heterogenous populations.

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Lack of Remuscularization Following Transplantation of Human Embryonic Stem Cell-Derived Cardiovascular Progenitor Cells in Infarcted Nonhuman Primates.

Rationale: Human pluripotent stem cell-derived cardiovascular progenitor cells (hPSC-CVPCs) should be thoroughly investigated in large animal studies before testing in clinical trials. Objective: To clarify if hPSC-CVPCs can engraft for long time in the heart of primates after myo-cardial infarction (MI) and compare the effectiveness and safety of immunosuppression with cy-closporine alone or multiple-drug regimen (MDR) containing cyclosporine, methylprednisolone, and Simulect in cynomolgous monkeys that had received intramyocardial injections of 1×107 EGFP-expressing hPSC-CVPCs after MI. A third group of animals received the immunosuppression MDR but without cell therapy after MI (MI+MDR group). Methods and Results: Measurements of EGFP gene levels and EGFP immunofluorescence staining indicated that the hPSC-CVPC engraftment rate was greater in the MI+MDR+CVPC group than that in the MI+Cyclosporine+CVPC group. However, even in the MI+MDR+CVPC group, no transplanted cells could be detected at 140 days after transplantation. Concomitantly, immunofluorescent analysis of CD3, CD4, and CD8 expression indicated that T lymphocyte infiltration in the CVPC-transplanted hearts was less in the MDR-treated animals than in the cyclosporine alone-treated animals. The re-covery of left-ventricular (LV) function at day 28 post-MI in the MI+MDR+CVPC group was better than that in the MI+MDR group. Apoptotic cardiac cells were also less common in the MI+MDR+CVPC group than in the MI+MDR group, while both immunosuppression regimens were associated with transient hepatic dysfunction. Conclusions: This is the largest study of hPSCs in non-human primates in cardiovascular field so far (n=32). Compared to cyclosporine alone, MDR attenuates immune rejection and improves survival of hPSC-CVPCs in primates; this is associated with less apoptosis of native cardiac cells and better re-covery of LV function at 28 days. However, even with MDR, transplanted hPSC-CVPCs do not en-graft and do not survive at 140 days after transplantation, thereby excluding remuscularization as a mechanism for the functional effect.

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Asymmetric Flow Field Flow Fractionation for the Characterization of Globule Size Distribution in Complex Formulations: A Cyclosporine Ophthalmic Emulsion Case.

Commonly used characterization techniques such as cryogenic-transmission electron microscopy (cryo-TEM) and batch-mode dynamic light scattering (DLS) are either time consuming or unable to offer high resolution to discern the poly-dispersity of complex drug products like cyclosporine ophthalmic emulsions. Here, a size-based separation and characterization method for globule size distribution using an asymmetric flow field flow fractionation (AF4) is reported for comparative assessment of cyclosporine ophthalmic emulsion drug products (model formulation) with a wide size span and poly-dispersity. Cyclosporine emulsion formulations that are qualitatively (Q1) and quantitatively (Q2) the same as Restasis® were prepared in house with varying manufacturing processes and analyzed using the optimized AF4 method. Based on our results, the commercially available cyclosporine ophthalmic emulsion, Restasis®, has a globule size span from 30 nm to a few hundred nanometers with majority smaller than 100 nm. The results with in-house formulations demonstrated the sensitivity of AF4 in determining the differences in the globule size distribution caused by the changes to the manufacturing process. It is concluded that the optimized AF4 is a potential analytical technique for comprehensive understanding of the microstructure and assessment of complex emulsion drug products with high poly-dispersity.

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Semifluorinated alkane based systems for enhanced corneal penetration of poorly soluble drugs.

Semifluorinated alkanes (SFAs) are amphiphilic liquids that can dissolve hydrophobic drugs to form clear solutions. This study evaluated the potential of two SFAs to act as vehicle for topical ocular drug delivery. After confirming ocular safety, an ex vivo corneal penetration model was developed to determine drug distribution and corneal bioavailability. Hydrophobic dye distribution in the different corneal layers was visualised under a confocal microscope. Corneal bioavailability of cyclosporine A (CsA) dissolved in perfluorobutylpentane (F4H5) or perfluorohexyloctane (F6H8) was compared to commercially available CsA ophthalmic emulsions, Restasis® and Ikervis®. Precorneal residence of the four test vehicles containing the hydrophobic dye was also compared using an ex vivo corneal tissue model. Preferential accumulation of the hydrophobic dye in the corneal epithelium was observed with higher amounts detectable when delivered via the SFAs compared to Restasis or Ikervis. A significant improvement in corneal CsA penetration was observed after application of a single dose of 0.05% CsA in F4H5 and F6H8 when compared to Restasis with the area under curve over 4 h (AUC(0 - 4 h)) being at least 8-fold greater for both SFAs (p<0.0001). Moreover, the AUC(0 - 4 h) of 0.1% CsA in F4H5 was almost 5-fold greater than Ikervis (p<0.0001). Finally, the precorneal residence time of both SFA solutions was significantly longer than that of the commercial emulsions with the AUC(0 - 60min) being 2- to 11-fold greater. This study demonstrated that SFAs can significantly improve the local bioavailability of hydrophobic drugs by increasing corneal penetration as well as prolonging precorneal residence. They therefore offer a promising new platform for topical drug delivery to the eye.

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