s2fieldsofstudy: Biology Medicine

text:
Leucine-Rich Repeat Kinase 2 (Lrrk2) Deficiency Diminishes the Development of Experimental Autoimmune Uveitis (EAU) and the Adaptive Immune Response

Background Mutations in LRRK2 are related to certain forms of Parkinson’s disease and, possibly, to the pathogenesis of Crohn’s disease. In both these diseases inflammatory processes participate in the pathogenic process. LRRK2 is expressed in lymphoid cells and, interestingly, Lrrk2 (-/-) mice were reported to develop more severe experimental colitis than their wild type (WT) controls. Here, we examined the possible involvement of LRRK2 in the pathogenesis of experimental autoimmune uveitis (EAU), an animal model for human uveitis, by testing Lrrk2 (-/-) mice for their capacity to develop this experimental eye disease and related immune responses. Methods Lrrk2 (-/-) mice and their WT controls (C57Bl/6) were immunized with interphotoreceptor retinoid-binding protein (IRBP) and compared for their development of EAU, delayed type hypersensitivity (DTH) by skin tests, production of cytokines in culture, and expression of interferon (IFN)-γ, interleukin (IL)-17 and FoxP3 by spleen cells, using flow cytometry. Peritoneal macrophages were examined for their production of cytokines/chemokines in culture following stimulation with LPS or the oligodeoxynucleotide CpG. The Lrrk2 (-/-) and WT mice were also compared for their response to bovine serum albumin (BSA). Results The Lrrk2 (-/-) mice developed lower levels of EAU, DTH responses and cytokine production by lymphocytes than did their WT controls. Intracellular expression of IFN-γ and IL-17, by spleen cells, and secretion of cytokines/chemokines by activated peritoneal macrophages of Lrrk2 (-/-) mice trended toward diminished levels, although variabilities were noted. The expression levels of FoxP3 by Lrrk2 (-/-) spleen cells, however, were similar to those seen in WT controls. Consistent with their low response to IRBP, Lrrk2 (-/-) mice responded to BSA less vigorously than their WT controls. Conclusions Lrrk2 deficiency in mice diminished the development of EAU and the related adaptive immune responses to IRBP as compared to the WT controls.

Introduction
Leucine-rich repeat kinase 2 (LRRK2), also known as Dardarin, plays an important role in the neural system; mutations in the LRRK2 gene are responsible for certain forms of Parkinson's disease [1][2][3]. In addition, mutations in the LRRK2 gene were found to be associated with Crohn's disease, an inflammatory bowel disease [4][5][6]. The function of LRRK2 in the CNS is not known, but examination of the pathogenic process of Parkinson's disease revealed the involvement of inflammatory processes in this condition, suggesting that a defect in the lymphoid system could play a role in the pathogenic process of this disease [7,8]. Immune-mediated inflammation is considered to be a major pathogenic mechanism of Crohn's disease [9,10].
Several published studies have provided evidence to show the involvement of LRRK2 in the immune system and accumulating data on this topic are summarized in an extensive recent review by Russo et al. [7]. The Lrrk2 gene and its protein have been detected in several cells involved in immunological and inflammatory processes, in particular B-cells, monocytes and dendritic cells [8,[11][12][13]. Furthermore, the expression of the LRRK2 protein and its gene were found to increase following exposure of these cells to microbial products and other pathogenic stimuli [11,12]. Deficiency in Lrrk2 in rats was found to perturbate the immunological homeostasis in rats [14]. he involvement of Lrrk2 in the pathogenic process of Crohn's disease was investigated in a study by Liu et al. [4], in which Lrrk2 deficient mice were found to be more susceptible than their wild type (WT) controls to experimental colitis, induced by treatment with dextran sulfate sodium.
In the present study we examined the effect of Lrrk2 deficiency on the susceptibility of mice to the induction of experimental autoimmune uveitis (EAU). EAU serves as an animal model for uveitic conditions in humans, a family of eye diseases, assumed to be immune-mediated, that includes sympathetic ophthalmia, "birdshot" chorioretinopathy, Behcet's disease, Vogt Koyanagi Harada (VKH) disease and sarcoidosis [15,16]. EAU is induced in mice by immunization with the retinal protein, interphotoreceptor retinoid-binding protein (IRBP). Unlike the observation with experimental colitis [1], mice deficient in Lrrk2 were found in our study to be less susceptible than the WT controls to the induction of EAU. In addition, the Lrrk2 (-/-) mice developed lower levels of cellular immunity against the immunizing antigen as compared to their WT controls.

Mice
Lrrk2 (-/-) mice were generated as described [17] and were further backcrossed onto the C57Bl/6J background, facilitated by genome scan. The deficiency of Lrrk2 in the deficient mice was determined by the absence of LRRK2 in tissue extracts, demonstrated by Western blotting (S1 Fig). Lrrk2 (-/-) mice and their WT littermates, or matched WT mice from the same colony, were used at 8-16 weeks of age. All experiments were done with the approval of the Animal Care and Use Committee of the National Eye Institute, NIH.

Induction and Evaluation of EAU
Lrrk2 (-/-) mice and their WT controls were immunized with bovine IRBP (150 μg) and human IRBP peptide 1-20 (200 μg), emulsified with complete Freund's adjuvant (CFA) containing 2.5 mg/ml killed Mycobacterium tuberculosis (DIFCO, Detroit, MI), administered subcutaneously in a volume of 0.2 ml. In addition, the mice were injected intraperitoneally with 0.2 μg pertussis toxin (List Biological Laboratories, Inc., Campbell, CA). The development of ocular inflammation was determined by histological examination on day 14 post-immunization, following euthanasia. Severity of disease, on a scale of 0-4, in half point increments, was scored as follows: focal non-granulomatous, monocytic infiltration in the choroid, ciliary body and retina were scored as 0.5. Retinal perivascular infiltration and monocytic infiltration in the vitreous were scored as 1. Granuloma formation in the uvea and retina, the presence of occluded retinal vasculitis, along with photoreceptor folds, serous detachment and loss of photoreceptors were scored as 2. In addition, retinal folding, the formation of granulomas at the level of the retinal pigmented epithelium and the development of subretinal neovascularization were scored as 3 and 4, according to the number of these pathological features.

Immunization with Bovine Serum Albumin (BSA)
Lrrk2 (-/-) mice and their WT controls were immunized with BSA (MP Biomedicals, Solon, OH), at 150 μg/mouse, emulsified with CFA. In addition, the mice were injected with pertussis toxin, at 0.2 μg/mouse. The immune responses against BSA of these mice were measured similarly to those for the mice immunized with IRBP, detailed below.

Delayed Type Hypersensitivity (DTH) Skin Tests
On day 12 post-immunization mice were skin-tested by injection of the immunizing antigen, IRBP or BSA, at 10 μg or 30 μg, respectively, into their ear pinnae, in a volume of 10 μl. The ear thickness was measured before injection and 48 hrs later, using a micrometer. The response was calculated by the equation: ear thickness at 48 hrs minus thickness pre-injection.

Intracellular Expression of IFN-γand IL-17 by Spleen Cells
Spleen cells from the immunized mice collected on day 14 post-immunization were incubated for 4 hrs with PMA at 20 ng/ml and ionomycin at 1μM (both from Sigma, St. Louis, MO) in the presence of GlogiStop (BD Bioscience, San Jose, CA). According to the manufacturer's instructions, the cells were then surface stained with antibody against CD4, conjugated with FITC and intracellular stained with APC-anti-IFN-γ and PE-anti-IL-17 (BD Bioscience, San Jose, CA). CD4 + cells were acquired on a MACS Quant analyzer (Miltenyi Biotec, San Diego, CA) and the data were analyzed by FlowJo software (FlowJo LLC., Ashland, OR).

Expression of FoxP3 by Spleen Cells
Spleen lymphocyte suspensions, prepared as described above were tested for their expression of FoxP3 using flow cytometry, following the manufacturer's protocol (eBioscience, San Diego, CA). In short, spleen cells were treated with the fixation/permeabilization buffer before intracellular staining with allophycocyanin-conjugated anti-FoxP3 antibody. Cells were acquired and data were analyzed as detailed above.

Statistical Methods
Statistical analysis was performed using GraphPad Prism 6 (GraphPad Software Inc. La Jolla, CA). Statistical significance was determined using unpaired t tests with p 0.05. Where nonnormal distributions were compared, the Mann-Whitney test was used. The pathological scores of the WT and the Lrrk2 (-/-) mouse groups (Fig 1A) represent the average score from each group in eight individual experiments. To allow comparison of ELISA results across multiple experiments ELISA results were normalized 0-1 with feature scaling, where:

Lrrk2 (-/-) Mice Develop Less Severe EAU than their WT Controls
We compared Lrrk2 (-/-) mice to their WT controls for their capacity to develop EAU when immunized with IRBP. The comparison was carried out in eight repeated experiments. The disease development was determined by histological examination and the accumulated data, summarized in Fig 1A, show that the pathological ocular changes in Lrrk2 (-/-) mice were lower than those of their WT controls. Similar to observations in other studies [18], C57Bl/6 mice vary in their susceptibility to EAU induction; yet, the difference between the accumulated EAU severity means of the deficient mice and their WT controls was significant (p = 0.0019) (Fig 1A). It is also noteworthy that in each of the eight repeated experiments, the severity means of pathological changes were lower in the deficient mice than in their corresponding WT controls. Fig 1B shows typical eye sections of an Lrrk2 (-/-) mouse and a WT control, demonstrating severe inflammation in the control mouse eye (severity score of 3.5+) and just moderate changes in the deficient mouse eye (severity score of 1.0+).
Lrrk2 (-/-) Mice Are Less Potent than their WT Controls in Development of DTH Response Similar to the pathogenic process of EAU, DTH responses are mediated by Th cells and we used this immunological assay to further compare between Lrrk2 (-/-) mice and their WT controls. The data of a representative experiment are shown in Fig 1C and demonstrate significantly stronger responses by the WT mice than by the Lrrk2 (-/-) mice, in line with the more severe EAU changes in the WT mice.

Proportions of Th1 and Th17 Cells in the Spleens of Immunized Lrrk2 (-/-) and WT Controls
Next, we determined the proportions of Th1 and Th17 cells in the spleens of the immunized mice, using flow cytometry to measure the proportions of cells intracellularly expressing IFNγor IL-17, respectively. Fig 2 records data of two experiments, similarly showing slightly higher levels of cells stained for the two signature antigens in the WT mice than in the Lrrk2 (-/-) ones.

Lymphocytes of Lrrk2 (-/-) Mice Produce Lower Levels of IFN-γ and IL-17 than do their WT Controls
Pathological changes of EAU have been shown to be mediated by Th1 and Th17 cells specific against IRBP [16,19]. To measure the specific immunological capacity of lymphocytes of the Due to variability among experiments, the differences between the Lrrk2 (-/-) and WT controls did not reach significance.

Proportions of FoxP3 Expressing Cells Are Similar in Spleens of Lrrk2 (-/-) and WT Control Mice
To examine the possibility that the inferior immune responsiveness in Lrrk2 (-/-) mice is due to increased levels of Treg cells, we compared by flow cytometry the proportions of CD4 cells expressing FoxP3 in spleens of the two mouse groups. FoxP3 is a transcription factor specifically expressed by Treg cells [20,21]. Fig 4A shows   Lrrk2 Deficiency Diminishes EAU Development

Comparison between Peritoneal Macrophages of Lrrk2 (-/-) and WT Control Mice for their Response to TLR Ligands in Culture
In addition to their participation in phagocytosis, macrophages play major roles in the immune system as producers of cytokines and chemokines. To compare between macrophages of Lrrk2 deficient mice and WT controls, we collected their induced peritoneal macrophages and measured the levels of certain cytokines and chemokines released in cultures stimulated with two TLR ligands, LPS and CpG [22]. The data collected in two separate experiments are combined in Fig 5 and show only small differences in levels of the majority of tested molecules produced by macrophages of the two mouse groups when stimulated by LPS or CpG. These observations are in line with findings of a study by Dzamko, et al [23]. It is of interest, however, that the mean levels of TNF-α were higher in supernatants of WT than of Lrrk2 (-/-) cells when cultured with either LPS or CpG.
The Immune Responsiveness of Lrrk2 (-/-) Mice to an Antigen other than IRBP, Namely BSA, is also Lower than that of the WT Controls To examine the specificity of the reduced immune response of Lrrk2 (-/-) mice we compared groups of Lrrk2 (-/-) and WT controls for their response to BSA. The immunized mice were tested for their DTH response and spleen cells were examined for their production of IFN-γ and IL-17 following exposure to BSA in culture. Data collected with these assays are recorded in Fig 6A and 6B and 6C, respectively, and show that, similar to their reduced response to IRBP, Lrrk2 (-/-) mice reacted to BSA with levels lower than those of their WT controls.

Discussion
Data recorded here show that the capacity of Lrrk2 (-/-) mice to develop EAU following immunization with IRBP is lower than that of their WT controls. This observation is in contrast to that of Liu et al [1], who found that Lrrk2 (-/-) mice developed more severe experimental colitis than their WT controls. The Lrrk2 (-/-) mice in the two studies were of the same colony, and the remarkable difference in their susceptibility to the two diseases could be mainly attributed to differences in the pathogenic mechanisms that mediate these two experimental diseases. EAU is initiated by the adaptive immune system [16,24], whereas innate immunity plays a role in the pathogenesis of experimental colitis [4,25,26]. In order to learn about possible mechanistic immunological differences between the Lrrk2 (-/-) mice and their WT controls, we compared the two groups of animals by a battery of immunological assays related to the pathogenic process of EAU. A correlation was observed between the susceptibility of mice to EAU induction and their DTH skin test response, another process mediated by Th cells. Both Th1 and Th17 are involved in the pathogenesis of EAU [16,19,27] and we examined these two populations in the Lrrk2 deficient mice and their WT controls by two assays, i.e., their proportions among the spleen lymphocytes in the immunized mice and their capacity to produce in culture their signature cytokines, IFN-γ and IL-17, respectively. The proportions of cells producing IFN-γ or IL-17, determined by flow cytometry, were found to be slightly higher in spleens of WT mice than in the Lrrk2 (-/-) mice. Production of cytokines by cultured spleen cells, measured by ELISA, revealed that lymphocytes from the Lrrk2 (-/-) mice were quite consistent in producing lower levels of IFN-γ as compared to their WT controls. The difference between the two groups of mice was less consistent for the release of IL-17, a finding that could be attributed to the production of this cytokine by non-Th17 cells, in particular γδT-cells and innate lymphoid cells [28]. It is conceivable that these non-Th17 cells are less affected by the Lrrk2 deficiency than Th17 cells.
Another family of cells involved in the immune response tested in this study were peritoneal macrophages. When stimulated in culture with LPS or CpG, macrophages from Lrrk2 (-/-) mice resembled those from the WT controls in their levels of production of the majority of tested cytokines and chemokines (Fig 5). It is of note that Liu et al. [4] found that bone marrow-derived macrophages from Lrrk2 (-/-) produced higher levels of several cytokines than did the WT control macrophages, when stimulated with Zymosan. The difference between the two studies could be due to different populations of macrophages used in the two studies and different stimuli; as shown by Liu et al, the patterns of cytokine release depends on the stimulus [4]. Interestingly, LRRK2 inhibition or deficiency was reported to attenuate the pro-inflammatory activities of microglia, the macrophage-like cells of the CNS, including their cytokine production [29,30].
Reduced immune response may be caused by an increase in proportion of Treg cells [20,21]. We tested this hypothetical situation by comparing the number of cells that express FoxP3 in the spleens of Lrrk2 (-/-) mice and WT controls. Essentially no differences were detected between the two mouse groups by this assay (Fig 4), indicating that the reduced immune responsiveness by Lrrk2 (-/-) mice cannot be attributed to increased activity of Treg cells.
Our finding that the Lrrk2 (-/-) mice also respond to BSA by levels lower than those of their WT controls further indicates that deficiency in Lrrk2 affects the adaptive immune response of the deficient mice. It is obviously of interest that the Lrrk2 (-/-) mice showed increased susceptibility to experimental colitis [4] and enhanced levels of innate immune response. Additional studies are needed to further investigate the impact of Lrrk2 deficiency on the immune response, particularly in view of the LRRK2 gene's involvement in the pathogenesis of Parkinson's disease, a condition in which inflammation plays an important role.


extfieldsofstudy: Medicine Biology

text:
BRCA1 185delAG Mutation Enhances Interleukin-1β Expression in Ovarian Surface Epithelial Cells

Familial history remains the strongest risk factor for developing ovarian cancer (OC) and is associated with germline BRCA1 mutations, such as the 185delAG founder mutation. We sought to determine whether normal human ovarian surface epithelial (OSE) cells expressing the BRCA1 185delAG mutant, BRAT, could promote an inflammatory phenotype by investigating its impact on expression of the proinflammatory cytokine, Interleukin-1β (IL-1β). Cultured OSE cells with and without BRAT were analyzed for differential target gene expression by real-time PCR, western blot, ELISA, luciferase reporter, and siRNA assays. We found that BRAT cells expressed increased cellular and secreted levels of active IL-1β. BRAT-expressing OSE cells exhibited 3-fold enhanced IL-1β mRNA expression, transcriptionally regulated, in part, through CREB sites within the (−1800) to (−900) region of its promoter. In addition to transcriptional regulation, BRAT-mediated IL-1β expression appears dualistic through enhanced inflammasome-mediated caspase-1 cleavage and activation of IL-1β. Further investigation is warranted to elucidate the molecular mechanism(s) of BRAT-mediated IL-1β expression since increased IL-1β expression may represent an early step contributing to OC.

Introduction
Ovarian cancer (OC), an inflammation associated cancer, is the deadliest gynecologic malignancy and is the 9th most common cancer among women [1]. Approximately 22,240 new cases are diagnosed and 14,030 women die of the disease annually [1]. Since most OC are diagnosed in late stage, the 5-year survival is approximately 27% [1].
Most OCs are epithelial ovarian carcinomas traditionally thought to arise from the ovarian surface epithelium (OSE) [2]; though more recently, studies have suggested that OC may arise from the fallopian tube epithelium [3,4]. The etiology of the disease is not completely understood, but family history (FH) is the strongest risk factor for the development of epithelial OC [5]. Hereditary OCs are often associated with mutation of the tumor suppressor breast cancer susceptibility gene 1 (BRCA1) [6]. Carriers of the BRCA1 mutation have a 30% risk of developing OC during their lifetime [7]. BRCA1 plays a role in DNA damage response, cell cycle signaling, recruitment of chromatin modifying proteins, interaction with transcription factors, and ubiquitin ligase activity [8]. Loss of these functions may contribute to the development of cancer by promoting genomic instability and accumulation of cancer-causing mutations. Mutation of the BRCA1 gene can also result in either "loss of function" or "gain of function" with appearance of novel truncated protein products, respectively (reviewed in [9]). Among possible gain-of-function BRCA1 mutations, the 185delAG mutation is one of the most common founder mutations and is associated with a 66% lifetime risk of developing OC [10].
The 185delAG BRCA1 truncated mutant, BRAT, is the result of a deletion of two nucleotides in the second exon of the BRCA1 gene leading to a reading frame shift and a premature stop codon at position 39. Previously, we 2 BioMed Research International demonstrated that human OSE cells with the BRAT mutation exhibited enhanced apoptosis and caspase-3 activation [11] as well as diminished levels of phosphorylated Akt, cellular inhibitor of apoptosis 1 (cIAP1), and X-linked inhibitor of apoptosis protein (XIAP) [12]. We also found that BRATmediated maspin expression was correlated with enhanced chemosensitivity [13] which is in agreement with clinical reports of increased survival in patients with elevated maspin levels [14]. Microscopic examination of ovarian specimens obtained following prophylactic oophorectomy from women with FH of OC indicated that greater than 85% presented with two or more abnormal OSE histologic features such as surface epithelial pseudostratification, surface papillomatosis, cortical invaginations of OSE, epithelial inclusion cysts, and epithelial hyperplasia [15]. Likewise, overtly normal OSE from women with a FH of breast and/or OC (FHOSE) in culture show an increased autonomy of the epithelial phenotype in terms of expression of the epithelial differentiation marker CA125 [16], persistence of an epithelial morphology [16], and reduction in epithelial-mesenchymal conversion as noted by the maintenance of high keratin expression, but a reduction of collagen type II expression compared with no family history (NFH) OSE [17]. Since OSE become more committed to an epithelial phenotype in the course of carcinogenesis, these reports coupled with our previous findings suggest that preneoplastic characteristics may already exist in overtly normal OSE in some women with a strong FH of breast and ovarian cancer.
Inflammation of the ovarian epithelium has also long been associated with increased risk for OC [18][19][20][21]. By promoting a local pelvic inflammatory reaction, endometriosis has been associated with increased risk for endometrioid adenocarcinoma and clear cell carcinoma of the ovary [22][23][24][25]. Likewise, chronic pelvic inflammatory disease, often resulting from infection, also supports a role for inflammation and increased risk for OC [26,27]. Lastly, epidemiological studies suggest that incessant ovulation causes rapid cycles of OSE division associated with repeated ovulatory traumatization and repair of the ovulatory defect [28]. Ultrastructural and histochemical studies of OSE in situ have shown that OSE migrates and proliferates extensively during repair of the OSE after ovulation [29,30]. During ovulatory repair, OSE is exposed to proinflammatory mediators such as cytokines, chemokines, matrix-remodeling enzymes, and various growth factors that can result in an increased risk for malignant transformation [31,32]. Reports of OC associated with ovarian hyperstimulation due to fertility drugs [33,34] further support a role for inflammatory traumatization in OC.
Interleukin-1 beta (IL-1 ) is a proinflammatory cytokine mainly produced by monocytes and macrophages [35], but which can also be produced by endothelial cells, fibroblasts, and epidermal cells in response to bacterial or innate immunity stimulation [36]. Interestingly, normal and malignant epithelial ovarian cells also produce IL-1 [2]. IL-1 is translated into a 31 kDa inactive precursor form that is cleaved intracellularly by caspase-1 into an active 17 kDa secreted form [35]. The aim of this study was to investigate the in vitro production of IL-1 in human OSE cell lines carrying the 185delAG BRCA1 mutation in order to determine whether enhanced IL-1 in these cells could contribute to an inflammatory phenotype.

Materials and Methods
2.1. Cell Culture and Transfection. The following SV 40-Large T-Antigen transfected human OSE (IOSE) cell lines were used: IOSE118 derived from a normal patient with a FH of breast and/or OC with wtBRCA1 status confirmed [13]; IOSE 121 derived from a normal patient with NFH of breast and/or OC though BRCA1 mutation status was not determined; and IOSE 3261-77 and IOSE 1816-686 derived from normal patients with a FH of breast and/or OC as well as confirmed carriers of the BRCA1 185delAG mutation. IOSE cells were cultured in Medium 199/MCDB 105 (Sigma, St. Louis, MO) with 10% fetal bovine serum (FBS) and gentamicin. Stable PCDNA (pcDNA3.1) and BRAT clones (BRATc1, BRATc2) were generated by transfection of 2.5 × 10 5 IOSE 118 cells with 2.5 g of pcDNA3.1 or Flag-BRAT with G418 resistance gene as previously described [13] using Program X-005, Kit V, and the Nucleofector device (Amaxa/Lonza, Walkersville, MD). Stable cells were maintained in 1 mg/mL G418 selection media and BRAT clones were confirmed to express BRAT by RT-PCR [13]. All cells were incubated at 37 ∘ C with 5% CO 2 . For knockdown studies, cells were cotransfected with 1.5 g ON-TARGETplus siRNA (siCREB, siCON) from Dharmacon (Chicago, IL). For treatment with IL-1 receptor antagonist (IL-1Ra), stable BRAT clones were washed with phosphate buffered saline (PBS) and media containing 0.1% FBS were added to the cells along with varying concentrations of IL-1Ra (R&D Systems, Inc., Minneapolis, MN). After 6 hours, the media were removed and RNA was extracted from the cells for PCR analyses as described below.

Western
Immunoblot. Cells were washed in PBS, trypsinized, pelleted, and washed 1-2 times in cold PBS. Cells were lysed for 30 minutes on ice in modified CHAPS buffer, and the lysate was centrifuged at 115,000 ×g, at 4 ∘ C for 1 hour. Then 30 g of protein was separated via 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to polyvinylidene fluoride (PVDF) membranes, dried, and blocked in 5% milk in Tween 20-Tris buffered saline. Blots were incubated in their respective primary antibodies overnight, followed by incubation with a horseradish peroxidase-(HRP-) conjugated secondary antibody (Fisher, Pittsburgh, PA), and developed via enhanced chemiluminescence substrate (ECL) (Pierce/Fisher, Pittsburgh, PA). Antibodies used

Enzyme-Linked Immunosorbant Assay.
For conditioned media (CM) analyses, medium containing 0.1% FBS was added to cells 24 hours after transfection/plating. After 24 hours, cells were counted and CM were collected and centrifuged to remove debris, aliquoted, and stored at (−80 ∘ C). For the IL-1 Enzyme-Linked Immunosorbant Assay (ELISA), CM were also concentrated 17x using Amicon Ultra-15 Centrifugal Filter Unit with Ultracel-10 membrane (Millipore, Billerica, MA) before storage at (−80 ∘ C). To assess the presence of mature IL-1 , an ELISA (R&D Systems, Inc., Minneapolis, MN) was performed on concentrated CM samples in triplicate according to the manufacturer's protocol. Fluorescence was read on an ELx800 Absorbance Microplate Reader (Biotek, Winooski, Vermont) using Gen5 Data Analysis Software (Biotek, Winooski, Vermont). Resultant values were derived from a standard curve and expressed as the mean IL-1 concentration of triplicate samples ± standard error. When cell viability varied significantly, IL-1 concentration was normalized to average cell number at time of CM collection.

Polymerase Chain
Reaction. RNA samples were isolated using TRIzol reagent from Invitrogen (Carlsbad, CA) as per the manufacturer's protocol and DNAse-treated.
For BRAT semiquantitative PCR to confirm transfection, 1 g total RNA, oligo(dT), and reverse transcriptase were used to generate single-strand cDNA as previously described [13]. The cDNA samples were amplified using the Applied Biosystems GeneAmp RNA PCR Core Kit (Foster City, CA). Primers used were Flag-BRAT sense (CGATGACAAAAT-GGATTTATCTGC), Flag-BRAT antisense (GAGACAGGT-TCCTTCATCAACTCC), actin sense (GGGAATTCAAAA-CTGGAACGGTGAAGG), and actin antisense (GGAAGC-TTATCAAAGTCCTCGGCCACA). The amplified products were separated by electrophoresis on a 10% polyacrylamide gel, stained with SYBR Green (Lonza, Rockland, ME), and photographed with the Kodak EDS 120 Digital Analysis System. The net intensity of each band was normalized to the respective endogenous control band.
For quantitative PCR, 100 ng total RNA was reversetranscribed to generate single-strand cDNA as previously described [13]. The cDNA samples were amplified in triplicate using Fast SYBR Green Master Mix (Applied Biosystems) on an Applied Biosystems Step One Plus instrument. Primers used were IL-1 sense (TCCAGGGACAGGATATG-GAG), IL-1 antisense (TCTTTCAACACGCAGGACAG), and actin (same as above). RQ (relative mean mRNA expression level) was calculated by the Step One software version 2.0. Using standard curves constructed for target and endogenous control genes, an arbitrary quantitative gene expression value was determined from the threshold cycle (Ct) for each gene for each sample. Target gene values were normalized to control gene values, and fold difference was determined by dividing by the designated reference/calibrator sample.
2.6. Immunoprecipitation. Cells from stable 118 pcDNA3.1 and BRAT clones were washed with PBS and incubated on ice with RIPA lysis buffer containing protease inhibitors. After 15 minutes, the cells were removed by scraping, the lysates were incubated for 60 minutes on ice, and then the lysates were centrifuged at 10,000 ×g for 10 minutes at 4 ∘ C. Two micrograms of primary antibody was incubated with 1 mg of whole cell lysate for 2 hours at 4 ∘ C. Protein A/G agarose suspension was added to lysate and antibody mixture followed by incubation at 4 ∘ C on a rocker overnight. The suspension was collected by centrifugation at 500 ×g for 2 minutes at 4 ∘ C. The pellet was washed with RIPA buffer containing protease inhibitors followed by analysis by western immunoblotting.

Statistics.
For real-time PCR, relative gene expression was expressed as the average fold change (RQ) of RQ min and RQ max calculated as 2 −(ΔΔCt+ ×SD(ΔCt)) and 2 −(ΔΔCt+ ×SD(ΔCt)) , respectively, at the 95% confidence interval, for 5 degrees of freedom and where SD = standard deviation, = confidence level and Ct = cycle threshold. For ELISA and reporter assays, Student's -tests were performed to assess statistical difference between means of triplicates ± standard error from three separate experiments.

IL-1 Levels Are Increased in IOSE Cells
Carrying the BRCA1 185delAG Mutation. To determine the relationship between the BRCA1 185delAG mutation and protein levels of IL-1 , we compared IL-1 protein levels in human OSE cell lines that endogenously carry this mutation to those with wild-type BRCA1. Both FH IOSE 3261-77 and 1816-686 cell lines with confirmed 185delAG mutation had 7-fold and ≥13-fold higher active IL-1 protein levels, respectively, as measured by western blot than the NFH cell line (IOSE-121) (Figure 1(a)). Likewise, 3261-77 and 1816-686 cell lines had 2-and 10-fold, respectively, higher levels of secreted IL-1 protein in their CM as measured by ELISA than the IOSE-121 cell line (Figure 1(b)). stably transfected with PCDNA or Flag-BRAT for IL-1 message and protein levels. BRATc1 had approximately 3fold higher levels of IL-1 RNA as measured by real-time PCR than the pcDNA3.1 cell line (Figure 2(a)). Furthermore, densitometric analyses indicated that 22% of cellular pro-IL-1 in BRAT cells was cleaved into its active form while only 5.5% of cellular pro-IL-1 in pcDNA3.1 cells was cleaved into its active form (Figure 2(b)). Lastly, BRATc1 and BRATc2 both had 2-fold and 1.5-fold, respectively, higher levels of secreted IL-1 protein levels as measured by ELISA than the pcDNA3.1 cells (Figure 2(c)).
To further evaluate BRAT-mediated IL-1 promoter activation, we performed an online transcription factor prediction analysis of the 900 bp promoter region identified earlier to determine potential transcription factor binding sites using the prediction software, PROMO [37,38]. Our analyses identified two potential CREB binding sites within the 900 bp promoter region. Further, densitometric analyses of western immunoblots performed for phospho-CREB and CREB levels revealed 30% increased levels of phospho-CREB in BRAT cells (Figure 3(b)). Based on this observation, we used pooled siRNA to knockdown CREB and western blot to confirm CREB silencing. We found that CREB knockdown resulted in partial loss of IL-1 promoter activity using the (−1800) reporter plasmid (Figure 3(c)).

Caspase-1, ASC, and NALP3 Protein Levels Are Increased in BRAT Cells.
To confirm intracellular cleavage of the inactive IL-1 into its active form, we measured caspase-1 levels, the known intracellular activator of IL-1 . Stably transfected pcDNA3.1 cells had about 3-fold more inactive precursor caspase-1 protein levels than BRAT cells as seen by western blot (Figure 4(a)). However, stably transfected BRAT cells showed up to a 50-fold increase in active caspase-1 by western blot (Figure 4(a)). Given that BRAT might increase IL-1 activation by caspase-1-mediated cleavage, we sought to determine if the NALP3-ASC inflammasome, which cleaves pro-caspase-1 into its active form, was enhanced in BRAT cells. Western immunoblots confirmed a 3-fold increase of ASC protein levels in BRAT cells compared to the pcDNA3.1 cells (Figure 4(b)). Likewise, immunoprecipitation followed by densitometric analyses showed that, when normalized to cellular protein, both ASC and NALP3-bound ASC protein levels were 44% and 250% higher, respectively, in BRAT cells (Figure 4(c)).

IL-1Ra Suppresses Proinflammatory Mediators in BRAT
Cells. To evaluate the potential autofeedback loop of IL-1 on OSE cells, we treated stably transfected BRAT-containing IOSE cells with the IL-1 receptor antagonist, IL-1Ra. Then, we measured mRNA expression levels of known IL-1 downstream transcription targets: IL-1 , IL-6, and IL-8 by realtime PCR. Treatment of BRAT cells with IL-1Ra resulted in up to a 50% loss of mRNA expression levels of IL-1 , IL-6, and IL-8 ( Figure 5).

Discussion
The results of the present study show that the 185delAG BRCA1 mutant protein, BRAt, increases IL-1 mRNA and protein levels in immortalized human OSE cell lines. Interestingly, compared to controls, increased levels of IL-1 varied among IOSE cell lines and may reflect differences in BRCA1 BioMed Research International 185delAG penetrance, BRAt expression, and/or wtBRCA1 expression [11,13]. Therefore, it is important to note that inherent differences of wtBrca1 expression among cell lines derived from patients endogenously carrying the 185delAG mutation can limit understanding the independent functions of BRAt in these cells. For these reasons, we transitioned our studies into transiently or stably transfected 185delAG cell lines in a confirmed wtBRCA1 background. We also demonstrated that BRAT-dependent expression is transcriptionally mediated, in part, via CREB binding sites. In addition, we found that BRAT-dependent activation of IL-1 protein by caspase-1 may be enhanced by increased expression of the NALP3-ASC inflammasome. While further investigation is required to discover the exact mechanism of enhanced NALP3-ASC protein expression in BRAT cells, we suspect that ASC expression may be CREB-dependent or AP1-dependent since the current and prior studies indicate BRAT-mediated gene expression through CREB and AP1 sites [39]. Further, CREB binding sites are likely present in the ASC promoter according to commercially available promoter ChIP assay (Qiagen, Valencia, CA) program analysis. Lastly, we confirmed that IL-1 plays a role in expression of proinflammatory mediators, IL-6 and IL-8, via the IL-1 receptor suggesting that BRAT might stimulate a proinflammatory environment that could promote OC oncogenesis.
Establishing animal and cell models of OC oncogenesis has proven to be difficult. Spontaneous development of OC in animal models occurs with a low frequency and highly variable phenotypes [40]. Furthermore, OC lacks a clear molecular profile making it difficult to elucidate a universal cause in OC initiation or progression [41][42][43]. Loss of some or all wild type functions of the BRCA1 gene product due to gene mutation is commonly associated with enhanced breast cancer and OC risk (reviewed in [9]). BRCA1 gene mutations resulting in a premature stop codon are generally susceptible to nonsense-mediated messenger RNA (mRNA) decay. However, two common risk-associated BRCA1 mutations, 185delAG and 5382InsC, were found to be unaffected by mRNA decay [44,45]. This study utilizes a stable OSE cell line model with intact endogenous biallele BRCA1 along with the 185delAG BRCA1 mutant transcript. By retaining a BRCA1 wild type background, we are able to conclude that changes observed are due to the presence of the 185delAG BRCA1 mutation and not due to the partial or complete loss of BRCA1. This is in keeping with previous studies that have shown other BRCA1 mutations with independent and novel gain-of-function roles in OC proliferation, chemosensitivity, tumorigenesis, and apoptosis [46,47]. Inflammation has long been suggested to contribute to tumor initiation, promotion, and progression [48]. Many components of the inflammatory pathway, including free radicals, cytokines, cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and vascular endothelial growth factor (VEGF) have been implicated in the development of various malignancies, including OC [49]. Immunohistochemical analyses revealed increased COX-2 expression in nonmucinous ovarian tumors and increased COX-2 expression correlating with poor prognosis [50]. Likewise, the present results are in keeping with others who reported a relation between proinflammatory mediators and increased OC risk. A case-control study by Clendenen et al. confirmed a positive association between circulatory inflammatory cytokines, IL-2, IL-4, IL-6, IL-12, and IL-13, and OC risk [51]. Also, increased serum levels of the inflammatory marker CRP have been associated with an increased risk of OC [20,52]. Yellapa et al. demonstrated increased expression of the proinflammatory cytokine, IL-16, in serum and tissue of early and late stage OC patients as well as increased IL-16 serum levels during the progression from normal to early and late stage OC progression in hens [53]. Further, since increasing IL-16 serum levels preceded detection of OC by transvaginal ultrasound, it is tempting to speculate that monitoring proinflammatory mediators, such as IL-1 , may signal disease in women at high risk for OC before detection by conventional means.
Epidemiological studies suggest that long-term usage of nonsteroidal anti-inflammatory drugs (NSAIDs) is associated with decreased OC risk [54][55][56]. Further, in vitro and in vivo use of phytochemicals [57] exert anti-inflammatory activity related to reduced OC progression by stabilizing  BioMed Research International p53 [58], enhancing cisplatin sensitivity [59], inhibiting cell growth and VEGF expression [60], and suppressing IL-6 function [61]. There are currently three IL-1 inhibitors used in the treatment of nonmalignant inflammatory conditions: recombinant IL-1Ra, anakinra; IL-1 trap, rilonacept; and monoclonal anti-IL-1 antibody, canakinumab [62]. Consequently, premalignant targeting of the proinflammatory mediator IL-1 and its upstream processors, caspase-1 and/or the NALP3 inflammasome, specifically in 185delAG BRCA1 mutation carriers, may reduce the risk of malignant transformation initiated by chronic inflammation. Lastly, tumors can develop within and in response to their inflammatory microenvironment. For example, Kim et al. observed localized IL-1 expression among normal, benign, and malignant canine mammary tumors such that IL-1 could not be detected in normal tissue while concentrated IL-1 expression was noted in the stroma of benign tumors and diffuse expression in malignant and metastatic tumors [63]. Similarly, IL-1 secreted by OC cells suppresses p53 expression in cancer-associated fibroblasts [64]. In OC, IL-1 also promotes invasiveness and tumor angiogenesis and induces immune suppression [65]. Constitutive production of IL-1 by human ovarian carcinoma cell lines [66] enhances their invasion capacities by increasing expression of matrix metalloproteinase-1 and stimulating production of proangiogenic factors [52,67]. Watanabe et al. showed that IL-1 produced by OC cells induced mesothelial cell beta1-integrindependent peritoneal metastasis [68]. Increased levels of IL-1 found in the serum and/or the ascites of OC patients have been associated with decreased survival [69]. Likewise, IL-1 can effect OC progression by altering the expression of other proinflammatory cytokines, such as IL-6 which is a downstream target of IL-1 . IL-6 is known to play a major role in OC progression and prognosis and may also be a potential marker of immunological and metabolic changes in OC [70]. Elevated levels of IL-6 have been found in the serum and ascites of OC patients [71] and are also associated with poor prognosis [2]. In support of the data reported herein, we recently found that urinary levels of IL-1 were generally 5 times greater in women with OC compared to healthy controls [72]. Interestingly, while exact BRCA1 mutational status could not be determined, we also found that urinary levels of IL-1 were 3 times higher among women with benign ovarian disease and a FH of breast/ovarian cancer compared with women with benign ovarian disease and lacking a FH of breast/ovarian cancer. Likewise, OC patients with a confirmed FH of cancer had urinary IL-1 levels several fold higher than OC patients without a known FH of cancer. Therefore, chronic presence of IL-1 within the ovarian microenvironment may enhance malignant transformation and underscores the complexity among cancer cells within their microenvironment for tumor progression.

Conclusions
Our data identify the proinflammatory mediator, IL-1 , as a novel target in OSE cells expressing the 185delAG BRCA1 mutation. Further, the mechanism by which BRAT regulates IL-1 expression is twofold: (1) CREB-dependent transcriptional control and (2) caspase-1 cleavage of pro-IL-1 . It is interesting to speculate that increases in IL-1B might be limited to 185delAG rather than common to multiple BRCA1 mutations. While we have not been able to study other BRCA1 mutations in similar detail, a recent study by Rebbeck et al. [73] suggests that differences in risk for breast and/or ovarian cancer may be linked to the locations of mutations in BRCA1 and BRCA2. Further, in prior publications, we have shown that targets downstream of 185delAG, including maspin, are unaffected when 185delAG is itself mutated to generate a further BRCA1 mutation [39] suggesting that the 185delAG BRCA1 mutation may promote tumorigenesis by a unique mechanism. Clearly, additional studies on the role of BRCA1 mutations to promote a chronic inflammatory phenotype are warranted since the potential also exists for IL-1 to serve as a predictor of OC and/or as a therapeutic target.


extfieldsofstudy: Biology Medicine

text:
Modulation of the antibiotic activity against multidrug resistant strains of 4-(phenylsulfonyl) morpholine

The compound 4-(Phenylsulfonyl) morpholine belongs to the class of sulfonamides, which are widely used in the treatment of a large number of diseases caused by microorganisms. This compound has a morpholine group, which is also known for its antimicrobial properties. The aim of the present study was to investigate the antimicrobial and modulating activity of 4-(Phenylsulfonyl) morpholine against standard and multi-resistant strains of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and strains of the fungi Candida albicans, C. tropicalis and C. krusei. Antimicrobial activity was assessed based on the minimum inhibitory concentration (MIC) using the microdilution method. MIC was ⩾1024 μg/mL for all microorganisms. Regarding modulating activity, the most representative effect occurred with the combination of 4-(Phenylsulfonyl) morpholine at a concentration of 128 μg/mL (MIC 1/8) and amikacin against P. aeruginosa 03, with a reduction in MIC from 312.5 to 39.06 μg/mL.

Introduction
Sulfonamides are substances with structures correlative to that of p-aminobenzoic acid. As competitive antagonists, sulfonamides impede their use by bacteria in the synthesis of folic acid, thereby affecting microorganisms that need to synthesize their own folic acid. As mammals do not synthesize folic acid, sulfonamides do not affect their metabolism (Alaburda et al., 2007). The importance of the sulfonamide nucleus is well established in pharmaceutical chemistry. New synthesized 5-substituted amino pyrazole sulfates in an attempt to find a therapeutic alternative for combating infection (Borges et al., 2004). A number of sulfonamides, especially those derived from p-aminobenzenesulfonamide, have structural variations that enhance their efficacy to obtain a greater action spectrum and increase their solubility in biologic systems (Coutinho et al., 2008a).
Morpholine derivatives constitute a new antifungal chemical group not correlated with other currently available medications with antifungal activity. These derivatives inhibit the biosynthesis of sterol by blocking two successive enzymatic processes: (1) inhibiting the biotransformation of lanosterol into zymosterol by blocking the enzyme C-14 sterol reductase and; (2) inhibiting the synthesis of ergosterol from the biotransformation of fecosterol into episterol by blocking the enzyme C-8 sterol isomerase; these enzymes are different from those inhibited by allylamines or azoles (Kerkenaar, 1987;Polak, 1988). The advantage in preparing morpholine derivatives resides in the fact that these compounds provide chlorhydrates that are soluble in water for pharmacological assays (Pinto et al., 2013). Different sulfonyl-hydrazones obtained from sulfonyl chloride exhibit anti-neoplasm, antibacterial, antinociceptive and other pharmacological activities against several and different targets (Oliveira, 2012). Synthetic substances have demonstrated efficacious antimicrobial action against resistant microorganisms. The determination of synergism or antagonism between antimicrobial agents is important to understanding the action mechanisms of these substances as well as resistance mechanisms. Moreover, in vivo and in vitro analyses are performed for the detection of therapeutic potential with the aim of finding alternative pathogen control methods (Cata˜o et al., 2010).
The aim of the present study was to investigate the antimicrobial and modulating activity of 4-(Phenylsulfonyl) morpholine against standard and multi-resistant strains of Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and strains of the fungi Candida albicans, Candida. tropicalis and Candida. krusei.

Microorganisms
The following strains were used: E. coli ATCC10536 and E. coli EC 27; S. aureus ATCC25923 and S. aureus SA358; P. aeruginosa ATCC15442 and P. aeruginosa PA; C. albicans ATCC40006; C. krusei ATCC6258 and C. tropicalis ATCC13803. Table 1 displays the resistance profile of the microorganisms. All strains were maintained on heart infusion agar (HIA, Difco Laboratories Ltd., San Diego, USA). Prior to the assays, the strains were cultivated in Brain-Heart Infusion broth (BHI, Difco Laboratories Ltd., San Diego, USA) for 18 h at 37°C.

Antimicrobial activity and antibiotic modulating activity
The minimum inhibitory concentration (MIC) of all microorganisms was determined in broth microdilution assays (CLSI, 2005) using an inoculum of 100 lL of each strain suspended in BHI broth at a concentration of 10 5 colony forming units/mL in 96-well microtitration plates, with dilutions in ½ series. An aliquot of 100 lL of 4-(Phenylsulfonyl) morpholine was added to each well. The final concentrations of the substance ranged from 512 to 8 lg/mL. The standard antibiotics (amikacin, gentamicin and neomycin) and antifungals (amphotericin B, benzoilmetronidazol, mebendazole and nystatin) were assayed at concentrations ranging from 512 to 8 lg/mL and were used as controls. The plates were incubated at 35°C for 24 h, after which the readings were performed with the aid of resazurin. The MICs were recorded as the least concentration necessary to the growth inhibition. For the assessment of the substance as a modulator of antibiotic and antifungal action, the MIC of antibiotics and antifungals was evaluated in the presence and absence of the substance in sterile microplates. The antibiotics and antifungals were analyzed at concentrations ranging from 512 to 0.5 lg/mL. All antibiotics tested were obtained from Sigma-Aldrich (St. Louis, USA). 4-(Phenylsulfonyl) morpholine was mixed in 10% BHI broth at sub-inhibitory concentrations, which were determined during the MIC evaluation tests. For the modulation test, the concentration of the extract solution was reduced eight fold (MIC/8). The preparation of the antibiotic solutions was performed with the addition of sterile distilled water at double concentration (1024 lg/mL) in relation to the initially defined concentration and volumes of 100 lL serially diluted (proportion: 1:1) in 10% BHI broth. Each well with 100 lL of the culture medium contained the diluted bacterial suspension (proportion: 1:10). The controls for the modulation assays were the same as those used in the MIC assays (Coutinho et al., 2008b). The plates were incubated at 35°C for 24 h, after which the reading was performed with the aid of resazurin. The antimicrobial assays were carried out in triplicate, with mean values used for the analysis.

Results and discussion
The MIC of the compound was >512 lg/mL for all bacterial (E. coli, S. aureus and P. aeruginosa) and fungal (C. albicans, C. krusei and C. tropicalis) strains, demonstrating a lack of clinical significance within the limits established by the protocol (Sriram et al., 2010). Moreover, a pilot test was performed using DMSO alone and no antimicrobial or modulating activity was found, indicating a lack of toxicity. Table 2 displays the effect of 4-(Phenylsulfonyl) morpholine on aminoglycoside activity, demonstrating modulation of the activity of amikacin and gentamicin, with a reduction in MIC against the Gram-negative strains. The most representative effect was obtained with the combination of 4-(Phenylsulfonyl) morpholine at a concentration of 128 lg/mL (MIC 1/8) and amikacin against PA 03, with a reduction in the MIC from 312.5 to 39.06 lg/mL. In contrast, the substance demonstrated no capacity to modulate the action of aminoglycosides against strains of S. aureus. These findings are in agreement with data reported in the literature testing synthetic products against bacteria for the reduction of microbial resistance (Sriram et al., 2010;Ravat et al., 2009;Zhanel et al., 2009).
The synergism found in the modulation of antibiotic activity against E. coli and P. aeruginosa may be explained by the fact that 4-(Phenylsulfonyl) morpholine is derived from sulfonamides. This class of antibiotics is represented in therapy by sulfamethoxazole, the action mechanism of which consists of the blocking of the enzyme dihydropteroate synthase in bacteria. Sulfonamides are bacteriostatic agents that act as antimetabolites of p-aminobenzoic acid, which is the substrate for bacterial dihydropteroate synthase, impeding the formation of the dihydropteroate and, consequently, the formation of N 5 ,N 10 -methylenetetrahydrofolate (Halland et al., 2014).
The morpholine group may also have affected the antimicrobial activity against E. coli and P. aeruginosa, as a study involving the combination of morpholine and dimorpholine demonstrated efficacy against bacteria and yeasts. The same study reports a significant decrease in fungal contamination (99.9%) and the degree of efficacy against this microorganism was maintained for a period of 28 days (Takahashi, 2012). In contrast, no significant results were achieved with regard to the strains of Candida tested.
The lack of modulating activity in neomycin against all strains may be explained by the difference in the structure of this aminoglycoside, which is a hydrophilic molecule composed of an aminocyclitol ring linked to one or more amino sugar through a glycosidic bond. In most of these compounds  Escherichia coli 27  Surgical wound AZT, AMI, AMP, CFR, KAN, CAZ, CCL, CF, CIP, CHL, IMI, SXT, TET, TOB  Escherichia coli ATCC10536  --Staphylococcus aureus 358  Surgical wound AMI, BUT, KAN, GEN, NEO, NET, OXA, PARA, SIS, TOB  Staphylococcus aureus ATCC25923 --Pseudomonas aeruginosa 03  Uroculture  AMI, CIP, CPM, CAZ, IMI,   with clinical usefulness, the aminocyclitol group is 2-deoxystreptamine, which can be substituted in the 4 and 5 or 4 and 6 position (Magnet and Blanchad, 2005), thereby influencing the solubility, polarity and absorption of the drug. Due to absorption to the intercellular space, a toxic effect is common to all aminoglycosides, except spectinomycin. Renal toxicity causes nerve damage, being the oto/neurotoxicity the main toxic effect of aminoglycosides (Vallejo et al., 2001;Oliveira et al., 2006).
Thus, the combination of compounds with aminoglycosides may be an option to minimize undesirable effects when used for the treatment of infection by E. coli and P. auruginosa, since such combinations have a synergic effect, with a considerable reduction in the MIC of these drugs, allowing the reduction of the essential dose necessary for treatment success (Figueredo et al., 2013).
The lack of a significant effect against S. aureus may be explained by the fact that this microorganism has a number of virulence mechanisms and a high degree of versatility regarding pathogenic strategies, which enhances its resistance to antibiotics (Coutinho et al., 2008a;Tavares, 2000). Epidemiological studies evaluating the resistance of S. aureus report mean resistance rates of 63.25%, 76.5%, 56% and 71.25% to ciprofloxacin, clindamycin, tetracycline and trimethoprim-sulfamethoxazole, respectively .
4-(Phenylsulfonyl) morpholine exhibited no significant modulating effect on the antifungal agents amphotericin B, benzoilmetronidazol, mebendazole and nystatin against C. albicans, C. tropicalis or C. krusei. The findings are in agreement with previous study demonstrating fungal resistance (Silva, 2009). This resistance may occur due to the presence of chitin in the cell wall of these microorganisms, which serves as structural support (Merzendorfer and Zimoch, 2003). As chitin is insoluble in most solvents, it hinders the entrance of the antifungal agent (Mathur and Narang, 1990).

Conclusion
The MIC of 4-(Phenylsulfonyl) morpholine was >512 lg/mL for all bacterial and fungal strains tested, demonstrating a lack of antimicrobial activity. However, when used as a modulating agent of amikacin and gentamicin, the synthetic substance tested lowered the MICs of these aminoglycosides against Gram-negative strains. This finding demonstrates that 4-(Phenylsulfonyl) morpholine has antibacterial and antibiotic modulatory activities, being this synthetic compound an interesting weapon against drug resistant bacteria. However, more data, mainly toxicological assays must be performed before this use.