Interleukin‐17A deficiency ameliorates streptozotocin‐induced diabetes

Interleukin-17 (IL-17) is a cytokine with critical functions in multiple autoimmune diseases. However, its roles in type I diabetes and the underlying mechanisms remain to be fully elucidated. In the current study, we investigated the impact of IL-17 deficiency on streptozotocin (STZ) -induced diabetes. Il-17^−/− mice exhibited attenuated hyperglycaemia and insulitis after STZ treatment compared with control mice. The Il-17^−/− mice had fewer CD8⁺ cells infiltrating the pancreas than wild-type controls after STZ injection. Wild-type mice showed increased percentage and number of splenic CD8⁺ cells and decreased Gr1⁺ CD11b⁺ myeloid-derived suppressor cells (MDSC) after STZ treatment, but Il-17^−/− mice maintained the percentages and numbers of splenic CD8⁺ cells and MDSC, suggesting that IL-17 is implicated in STZ-induced cellular immune responses in the spleen. We further purified the MDSC from spleens of STZ-treated mice. Il-17^−/− MDSC showed increased ability to suppress CD8⁺ cell proliferation in vitro compared with wild-type MDSC. Transfer of MDSC to diabetic mice showed that MDSC from Il-17^−/− mice could ameliorate hyperglycaemia. Moreover, recipients with MDSC from Il-17^−/− mice had a decreased percentage of CD8⁺ cell in the spleen compared with recipients with MDSC from wild-type mice. These data suggest that IL-17 is required in splenic MDSC function after STZ delivery. In summary, our study has revealed a pathogenic role of IL-17 in an STZ-induced diabetes model with important implications for our understanding of IL-17 function in autoimmune diseases.     

Granulocyte‐macrophage colony‐stimulating factor,a potent adjuvant for polarization to Th‐17 pattern: an experience on HIV‐1 vaccine model

Cytokines are mediators for polarization of immune response in vaccines. Studies show that co‐immunization of DNA vaccines with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) can increase immune responses. Here, experimental mice were immunized with HIV‐1_{tat/pol/gag/env} DNA vaccine with GM‐CSF and boosted with recombinant vaccine. Lymphocyte proliferation with Brdu and CTL activity, IL‐4, IFN‐γ, IL‐17 cytokines, total antibody, and IgG1 and IgG2a isotypes were assessed with ELISA. Results show that GM‐CSF as adjuvant in DNA immunization significantly increased lymphocyte proliferation and IFN‐γ cytokines, but CTL response was tiny increased. Also GM‐CSF as adjuvant decreased IL‐4 cytokine vs mere vaccine group. IL‐17 in the group that immunized with mixture of DNA vaccine/GM‐CSF was significantly increased vs DNA vaccine group. Result of total antibody shows that GM‐CSF increased antibody response in which both IgG1 and IgG2a increased. Overall, results confirmed the beneficial effect of GM‐CSF as adjuvant to increase vaccine immunogenicity. The hallmark result of this study was to increase IL‐17 cytokine with DNA vaccine/GM‐CSF immunized group. This study for the first time provides the evidence of the potency of GM‐CSF in the induction of IL‐17 in response to a vaccine, which is important for control of infection such as HIV‐1.     

MiR‐505 promotes M2 polarization in choroidal neovascularization model mice by targeting transmembrane protein 229B

We aimed to analyse the relative abundance of miR‐505 in age‐related macular degeneration (AMD) and elucidate its underlying mechanisms. Relative expression of miR‐505 was analysed by real‐time polymerase chain reaction (PCR). Macrophage polarization was characterized by measurement of molecular markers including Ym‐1, Arg‐1, TNF‐α and iNOS via both real‐time PCR and Western blot. Vascular endothelial growth factor (VEGF) content was determined by enzyme‐linked immunosorbent assay. Choroidal neovascularization (CNV) formation was evaluated by choroidal flat mount technique. The regulatory action of miR‐505‐5p on 3′UTR of Transmembrane Protein 229B (TMEM229B) was interrogated by luciferase reporter assay. miR‐505 was aberrantly upregulated in both AMD and laser‐induced choroidal neovascularization mouse model. Administration with miR‐505 specific inhibitor suppressed M2 polarization in CNV mice as indicated by decreasing both Ym‐1 and Arg‐1. Meanwhile, VEGF expression and CNV formation were greatly suppressed by miR‐505 inhibition as well. The similar phenotype was consolidated in Prostaglandin E2 (PGE2)‐stimulated bone marrow‐derived macrophages. At the molecular level, miR‐505‐5p directly targeted and negatively regulated TMEM229B expression, while forced ectopic expression of TMEM229B significantly rescued miR‐505‐imposed M2 polarization. Our data have uncovered the critical contribution of miR‐505 in AMD, which is predominantly mediated by downregulation of TMEM229B.     

Interleukin‐17A participates in podocyte injury by inducing IL‐1β secretion through ROS‐NLRP3 inflammasome‐caspase‐1 pathway

Studies show that the Th17/IL ‐17A axis plays an important role in the pathogenesis of kidney diseases. Previously, we also showed that IL ‐17A may play a role in the pathogenesis of primary nephrotic syndrome; however, the underlying mechanism(s) is unclear. The aim of this study was to explore the molecular mechanism of IL ‐17A‐inducing podocyte injury in vitro. In this study, the NLRP 3 inflammasome activation and the morphology of podocytes were detected by Western blot and immunofluorescence. The results showed that podocytes persistently expressed IL ‐17A receptor and that NLRP 3 inflammasome in these cells was activated upon exposure to IL ‐17A. Also, activity of caspase‐1 and secretion of IL ‐1β increased in the presence of IL ‐17A. In addition, IL ‐17A disrupted podocyte morphology by decreasing expression of podocin and increasing expression of desmin. Blockade of intracellular ROS or inhibition of caspase‐1 prevented activation of the NLRP 3 inflammasome, thereby restoring podocyte morphology. Taken together, the results suggest that IL ‐17A induces podocyte injury by activating the NLRP 3 inflammasome and IL ‐1β secretion and contributes to disruption of the kidney's filtration system.     

M2 polarization of murine peritoneal macrophages induces regulatory cytokine production and suppresses T‐cell proliferation

Bone‐marrow‐derived macrophages are divided into two phenotypically and functionally distinct subsets, M1 and M2 macrophages. Recently, it was shown that adoptive transfer of M2‐polarized peritoneal macrophages reduced the severity of experimental colitis in mice. However, it is still unclear whether peritoneal macrophages possess the same ability to be polarized to cells with functionally different phenotypes and cytokine production patterns as bone‐marrow‐derived macrophages. To address this question, we examined the ability of peritoneal macrophages to be polarized to the M1 and M2 phenotypes and determined the specific cytokine profiles of cells with each phenotype. We showed that peritoneal macrophages, as well as bone‐marrow‐derived macrophages, were differentiated into M1 and M2 phenotypes following stimulation with interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4)/IL‐13, respectively. Following in vitro stimulation with lipopolysaccharide, M2‐polarized peritoneal macrophages predominantly expressed T helper type 2 (Th2) cytokines and regulatory cytokines, including IL‐4, IL‐13, transforming growth factor‐β and IL‐10, whereas M1‐polarized peritoneal macrophages expressed negligible amounts of Th1 and pro‐inflammatory cytokines. ELISA showed that M2‐polarized peritoneal macrophages produced significantly more IL‐10 than M1‐polarized peritoneal macrophages. Notably, M2‐polarized peritoneal macrophages contributed more to the suppression of T‐cell proliferation than did M1‐polarized peritoneal macrophages. The mRNA expression of Th2 cytokines, including IL‐4 and IL‐13, increased in T‐cells co‐cultured with M2‐polarized macrophages. Hence, our findings showed that M2 polarization of peritoneal macrophages induced regulatory cytokine production and suppressed T‐cell proliferation in vitro, and that resident peritoneal macrophages could be used as a new adoptive transfer therapy for autoimmune/inflammatory diseases after polarization to the regulatory phenotype ex vivo.     

miR-210通过MMP-2下调M2/M1巨噬细胞比例抑制肺癌细胞生长

目的研究miR-210通过MMP-2调控M2/M1巨噬细胞比例对肺癌细胞生长的影响及对相关机制进行初步探讨。方法将小鼠Lewis lung carcinoma (LLC)-1细胞皮下植入C57BL/6小鼠构建小鼠肺癌模型,荧光定量PCR检测miR-210表达。进一步取40只肺癌建模成功的小鼠,并随机分为4组:对照组小鼠尾静脉注射AAV9-Fugw腺相关病毒;AAV9-OE-miR-210组小鼠尾静脉注射AAV9-OE-miR-210腺相关病毒; AAV9-OE-MMP-2组小鼠尾静脉注射AAV9-OE-MMP-2腺相关病毒;AAV9-OE-miR-210+AAV9-OE-MMP-2组小鼠尾静脉注射AAV9-OE-miR-210和AAV9-OE-MMP-2腺相关病毒。所有小鼠注射腺相关病毒滴度为108TU,病毒注射时间为4周。检测4组小鼠肿瘤大小变化,WGA染色检测4组小鼠肺癌细胞大小变化。另外,取生长融合至80%的LLC1细胞,并随机分为2组:WT组转染包含野生型MMP-2 3’-UTR的构建物,Mut组转染突变型MMP-2 3’-UTR的构建物。细胞转染48 h后,荧光素酶实验检测m...     

Interleukin‐17A: a unique pathway in immune‐mediated diseases: psoriasis,psoriatic arthritis and rheumatoid arthritis

Experimental evidence points to the importance of the cytokine interleukin‐17A (IL‐17A) in the pathogenesis of several immunoinflammatory diseases including psoriasis, psoriatic arthritis and rheumatoid arthritis. Although a principal effector of T helper type 17 cells, IL‐17A is produced by many other cell types including CD8⁺ T cells and γδ T cells, and is found at high levels associated with mast cells and neutrophils at sites of skin and joint disease in humans. IL‐17A up‐regulates expression of numerous inflammation‐related genes in target cells such as keratinocytes and fibroblasts, leading to increased production of chemokines, cytokines, antimicrobial peptides and other mediators that contribute to clinical disease features. Importantly, IL‐17A must be considered within the context of the local microenvironment, because it acts synergistically or additively with other pro‐inflammatory cytokines, including tumour necrosis factor. Several direct IL‐17A inhibitors have shown promising activity in proof of concept and phase 2 clinical studies, thereby providing confirmation of experimental data supporting IL‐17A in disease pathogenesis, although levels of response are not predicted by pre‐clinical findings. IL‐17A inhibitors produced rapid down‐regulation of the psoriasis gene signature and high clinical response rates in patients with moderate‐to‐severe plaque psoriasis, consistent with an important role for IL‐17A in psoriasis pathogenesis. Clinical response rates with IL‐17A inhibitors in psoriatic arthritis and rheumatoid arthritis, however, were improved to a lesser degree compared with placebo, suggesting that IL‐17A is either important in a subset of patients or plays a relatively minor role in inflammatory joint disease. Ongoing phase 3 clinical trials should provide further information on the role of IL‐17A in these diseases.     

Interleukin‐17‐producing γδ+ T cells protect NOD mice from type 1 diabetes through a mechanism involving transforming growth factor‐β

Whether interleukin (IL)‐17 promotes a diabetogenic response remains unclear. Here we examined the effects of neutralization of IL‐17 on the progress of adoptively transferred diabetes. IL‐17‐producing cells in non‐obese diabetic (NOD) mice were identified and their role in the pathogenesis of diabetes examined using transfer and co‐transfer assays. Unexpectedly, we found that in vivo neutralization of IL‐17 did not protect NOD–severe combined immunodeficiency (SCID) mice against diabetes transferred by diabetic splenocytes. In NOD mice, γδ⁺ T cells were dominated by IL‐17‐producing cells and were found to be the major source of IL‐17. Interestingly, these IL‐17‐producing γδ T cells did not exacerbate diabetes in an adoptive transfer model, but had a regulatory effect, protecting NOD mice from diabetes by up‐regulating transforming growth factor (TGF)‐β production. Our data suggest that the presence of IL‐17 did not increase the chance of the development of diabetes; γδ T cells protected NOD mice from diabetes in a TGF‐β‐dependent manner, irrespective of their role as major IL‐17 producers.     

Vasoactive intestinal peptide suppresses macrophage‐mediated inflammation by downregulating interleukin‐17A expression via PKA‐ and PKC‐dependent pathways

Interleukin (IL)‐17A is a pro‐inflammatory cytokine that markedly enhances inflammatory responses in the lungs by recruiting neutrophils and interacting with other pro‐inflammatory mediators. Reducing the expression of IL‐17A could attenuate inflammation in the lungs. However, whether VIP exerts its anti‐inflammatory effects by regulating the expression of IL‐17A has remained unclear. Here, we show that there is a remarkable increase of IL‐17A in bronchoalveolar lavage fluid (BALF) and lung tissue of mice with acute lung injury (ALI). Moreover, lipopolysaccharides (LPS) stimulated elevated expression of IL‐17A, which was evident by the enhanced levels of mRNA and protein observed. Furthermore, we also found that VIP inhibited LPS‐mediated IL‐17A expression in a time‐ and dose‐dependent manner in an in vitro model of ALI and that this process might be mediated via the phosphokinase A (PKA) and phosphokinase C (PKC) pathways. Taken together, our results demonstrated that VIP might be an effective protector during ALI by suppressing IL‐17A expression.     

Interleukin‐21 (IL‐21) synergizes with IL‐2 to enhance T‐cell receptor‐induced human T‐cell proliferation and counteracts IL‐2/transforming growth factor‐β‐induced regulatory T‐cell development

Interleukin‐2 (IL‐2) is a mainstay for current immunotherapeutic protocols but its usefulness in patients is reduced by severe toxicities and because IL‐2 facilitates regulatory T (Treg) cell development. IL‐21 is a type I cytokine acting as a potent T‐cell co‐mitogen but less efficient than IL‐2 in sustaining T‐cell proliferation. Using various in vitro models for T‐cell receptor (TCR)‐dependent human T‐cell proliferation, we found that IL‐21 synergized with IL‐2 to make CD4⁺ and CD8⁺ T cells attain a level of expansion that was impossible to obtain with IL‐2 alone. Synergy was mostly evident in naive CD4⁺ cells. IL‐2 and tumour‐released transforming growth factor‐β (TGF‐β) are the main environmental cues that cooperate in Treg cell induction in tumour patients. Interleukin‐21 hampered Treg cell expansion induced by IL‐2/TGF‐β combination in naive CD4⁺ cells by facilitating non‐Treg over Treg cell proliferation from the early phases of cell activation. Conversely, IL‐21 did not modulate the conversion of naive activated CD4⁺ cells into Treg cells in the absence of cell division. Treg cell reduction was related to persistent activation of Stat3, a negative regulator of Treg cells associated with down‐modulation of IL‐2/TGF‐β‐induced phosphorylation of Smad2/3, a positive regulator of Treg cells. In contrast to previous studies, IL‐21 was completely ineffective in counteracting the suppressive activity of Treg cells on naive and memory, CD4⁺ and CD8⁺ T cells. Present data provide proof‐of‐concept for evaluating a combinatorial approach that would reduce the IL‐2 needed to sustain T‐cell proliferation efficiently, thereby reducing toxicity and controlling a tolerizing mechanism responsible for the contraction of the T‐cell response.     

Epstein–Barr virus‐induced gene 3 suppresses T helper type 1, type 17 and type 2 immune responses after Trypanosoma cruzi infection and inhibits parasite replication by interfering with alternative macrophage activation

The Epstein–Barr virus‐induced gene 3 (EBI3) is a member of the interleukin‐12 (IL)‐12) family structurally related to the subunit p40 of IL‐12 and forms a heterodimer either with the p28 subunit to build IL‐27 or with p35 to form IL‐35. Interleukin‐27 is secreted by antigen‐presenting cells whereas IL‐35 appears to be produced mainly by regulatory T cells and regulatory B cells but both cytokines negatively regulate inflammatory immune responses. We here analysed the function of EBI3 during infection with the intracellular parasite Trypanosoma cruzi. Compared with C57BL/6 wild‐type mice, EBI3‐deficient (EBI3^?/?) mice showed a higher parasitaemia associated with an increased mortality rate. The EBI3^?/? mice displayed an elevated inflammatory immune response with an increased production of T helper type 1 (Th1‐), Th2‐ and Th17‐derived cytokines. The increased Th2 immune response appears to have over‐ridden the otherwise protective Th1 and Th17 immune responses by the induction of arginase‐1‐expressing alternatively activated macrophages in these mice. Hence, neutralization of IL‐4 and arginase‐1 activity partially restored protective immune responses in EBI3^?/? mice. So far, our results demonstrate that EBI3 is an essential general regulator of inflammatory immune responses in experimental Chagas disease and is required for control of T. cruzi infection by inhibiting Th2‐dependent alternative macrophage activation. Further studies are needed to dissect the underlying mechanisms and clarify whether EBI3 association with IL‐27 or/and IL‐35 accounts for its anti‐inflammatory character in parasitic disease.     

Synergistic effect of interleukin‐17 and tumour necrosis factor‐α on inflammatory response in hepatocytes through interleukin‐6‐dependent and independent pathways

The proinflammatory cytokines interleukin (IL)‐17 and tumour necrosis factor (TNF)‐α are targets for treatment in many chronic inflammatory diseases. Here, we examined their role in liver inflammatory response compared to that of IL‐6. Human hepatoma cells (HepaRG, Huh7.5 and HepG2 cells) and primary human hepatocytes (PHH) were cultured with IL‐6, IL‐17 and/or TNF‐α. To determine the contribution of the IL‐6 pathway in the IL‐17/TNF‐α‐mediated effect, an anti‐IL‐6 receptor antibody was used. IL‐17 and TNF‐α increased in synergy IL‐6 secretion by HepaRG cells and PHH but not by Huh7.5 and HepG2 cells. This IL‐17/TNF‐α synergistic cooperation enhanced the levels of C‐reactive protein (CRP) and aspartate aminotransferase (ASAT) in HepaRG cell and PHH cultures through the induction of IL‐6. IL‐17/TNF‐α also up‐regulated IL‐8, monocyte chemoattractant protein (MCP)‐1 and chemokine (C‐C motif) ligand 20 (CCL20) chemokines in synergy through an IL‐6‐independent pathway. Interestingly, first exposure to IL‐17, but not to TNF‐α, was crucial for the initiation of the IL‐17/TNF‐α synergistic effect on IL‐6 and IL‐8 production. In HepaRG cells, IL‐17 enhanced IL‐6 mRNA stability resulting in increased IL‐6 protein levels. The IL‐17A/TNF‐α synergistic effect on IL‐6 and IL‐8 induction was mediated through the activation of extracellular signal‐regulated kinase (ERK)‐mitogen‐activated protein kinase, nuclear factor‐κB and/or protein kinase B (Akt)–phosphatidylinositol 3‐kinase signalling pathways. Therefore, the IL‐17/TNF‐α synergistic interaction mediates systemic inflammation and cell damage in hepatocytes mainly through IL‐6 for CRP and ASAT induction. Independently of IL‐6, the IL‐17A/TNF‐α combination may also induce immune cell recruitment by chemokine up‐regulation. IL‐17 and/or TNF‐α neutralization can be a promising therapeutic strategy to control both systemic inflammation and liver cell attraction.     

Interleukin‐17‐ and interleukin‐22‐secreting myelin‐specific CD4+ T cells resistant to corticoids are related with active brain lesions in multiple sclerosis patients

Multiple sclerosis (MS) is thought to be an autoimmune disorder. It is believed that immunological events in the early stages have great impact on the disease course. Therefore, we aimed to evaluate the cytokine profile of myelin basic protein (MBP)‐specific T cells from MS patients in the early phase of the disease and correlate it to clinical parameters, as well as to the effect of in vitro corticoid treatment. Peripheral T cells from MS patients were stimulated with MBP with our without hydrocortisone for 5 days. The cytokines level were determined by ELISA. The number of active brain lesions was determined by MRI scans, and the neurological disabilities were assessed by Expanded Disability Status Scale scores. Our results demonstrated that MS‐derived T cells responded to MBP by producing high levels of T helper type 1 (Th1) and Th17 cytokines. Although the production of interleukin‐6 (IL‐6), granulocyte–macrophage colony‐stimulating factor, IL‐17 and IL‐22 was less sensitive to hydrocortisone inhibition, only IL‐17 and IL‐22 levels correlated with active brain lesions. The ability of hydrocortisone to inhibit IL‐17 and IL‐22 production by MBP‐specific CD4⁺ T cells was inversely related to the number of active brain lesions. Finally, the production of both cytokines was significantly higher in cell cultures from Afrodescendant patients and it was less sensitive to hydrocortisone inhibition. In summary, our data suggest that IL‐17‐ and IL‐22‐secreting CD4⁺ T cells resistant to corticoids are associated with radiological activity of the MS in early stages of the disease, mainly among Afrodescendant patients who, normally, have worse prognosis.     

M1/M2型巨噬细胞极化参与肾组织炎症损伤和修复进程

目的:研究M1/M2型巨噬细胞在肾组织炎症损伤和修复进程中的分布趋势及作用。方法:将45只雄性SD大鼠随机分为缺血再灌注损伤(IRI)组和单侧输尿管梗阻(UUO)组两部分。IRI组分为假手术(sham)组及术后0、6、24和72 h组;UUO组分为sham组及梗阻3、7和14 d组;每个时相5只大鼠。全自动生化分析仪检测血肌酐和尿素氮水平;HE与免疫组织化学染色分别观察肾组织损伤及CD68的表达;流式细胞术检测M1(CD68+、F4/80+和CD16/32+)和M2(CD68+、F4/80+和CD206+)型巨噬细胞的分布;ELISA检测诱导型一氧化氮合酶(i NOS)、精氨酸酶1(Arg-1)、转化生长因子β1(TGF-β1)及肿瘤坏死因子α(TNF-α)水平。结果:IRI早期,肾组织损伤程度随再灌时间延长而加剧,并与CD68+巨噬细胞浸润呈一致性变化趋势;至24 h,组织损伤与巨噬细胞浸润最为严重;但之后随时间延长,损伤和巨噬细胞浸润反而减轻。UUO组中,肾损伤随梗阻时间延长而加剧,14d时纤维化明显;而巨噬细胞浸润第7天最为严重,之后又有减轻。流式细胞术分析显示IRI和UUO损伤早期浸润的巨噬细胞以M1型为主,i NOS表达较高;IRI和UUO损伤后期巨噬细胞均向M2型极化,Arg-1水平明显增加;巨噬细胞这种变化趋势与肾组织在不同时间节点出现损伤与修复表征存在明显相关性。深入分析发现,M1型巨噬细胞可通过诱导TNF-α高表达促进早期炎症损伤,M2型巨噬细胞通过提高TGF-β1水平促进后期纤维增生性修复。结论:UUO和IRI过程中均存在巨噬细胞极化:M1型巨噬细胞可诱导早期损伤,M2型巨噬细胞则参与损伤后期的纤维性修复。M1和M2型巨噬细胞在损伤修复过程中的极化特征可为临床治疗提供指导。     

Peritumoral brain edema in angiomatous supratentorial meningiomas: an investigation of the vascular endothelial growth factor A pathway

The aim of this work was to study the vascular endothelial growth factor A (VEGF‐A) pathway and peritumoral brain edema (PTBE) through comparison of non‐angiomatous and angiomatous meningiomas. Meningiomas are common intracranial tumors, which often have PTBE. VEGF‐A is an integral part of PTBE formation and angiogenesis, and the capillary‐rich angiomatous meningiomas are known for their PTBE. The VEGF‐A receptor VEGFR‐2 is responsible for the angiogenic effect of VEGF‐A on endothelial cells, which is enhanced by the co‐receptor neuropilin‐1. Forty non‐angiomatous, 22 angiomatous meningiomas, and 10 control tissue samples were collected for the study. Magnetic resonance images were available for 40 non‐angiomatous and 10 angiomatous meningiomas. Tissue sections were immunostained for CD34, MIB‐1, estrogen‐ and progesterone receptors. ELISA, chemiluminescence, and RT‐qPCR were used for VEGF‐A, VEGFR‐2, and neuropilin‐1 protein and mRNA quantification. Angiomatous meningiomas had larger PTBE (695 vs 218 cm³, p = 0.0045) and longer capillary length (3614 vs 605 mm/mm³, p < 0.0001). VEGF‐A mRNA, neuropilin‐1 mRNA, and VEGFR‐2 protein levels were higher in angiomatous meningiomas independently of the capillary length (p < 0.05). Neuropilin‐1 protein levels were lower in angiomatous meningiomas (p < 0.0001). The VEGF‐A pathway and tumor capillary length may be essential for PTBE‐formation in meningiomas. Further investigations of this pathway could lead to earlier therapy and targeted pharmacological treatment options.