Validated international definition of the thrombocytopenia, anasarca, fever, reticulin fibrosis, renal insufficiency, and organomegaly clinical subtype (TAFRO) of idiopathic multicentric Castleman disease.

Thrombocytopenia, anasarca, fever, reticulin fibrosis, renal insufficiency, and organomegaly (TAFRO) syndrome is a heterogeneous entity manifesting with a constellation of symptoms described above that can occur in the context of idiopathic multicentric Castleman disease (iMCD) as well as infectious diseases, malignancies, and rheumatologic disorders. So, iMCD-TAFRO is an aggressive subtype of iMCD with TAFRO syndrome and often hyper-vascularized lymph nodes. Since we proposed diagnostic criteria of iMCD-TAFRO in 2016, we have accumulated new insights on the disorder and additional cases have been reported worldwide. In this systematic review and cohort analysis, we established and validated a definition for iMCD-TAFRO. First, we searched PubMed and Japan Medical Abstracts Society databases using the keyword "TAFRO" to extract cases. Patients with possible systemic autoimmune diseases and hematologic malignancies were excluded. Our search identified 54 cases from 50 articles. We classified cases into three categories: (1) iMCD-TAFRO (TAFRO syndrome with lymph node histopathology consistent with iMCD), (2) possible iMCD-TAFRO (TAFRO syndrome with no lymph node biopsy performed and no other co-morbidities), and (3) TAFRO without iMCD or other co-morbidities (TAFRO syndrome with lymph node histopathology not consistent with iMCD or other comorbidities). Based on the findings, we propose an international definition requiring four clinical criteria (thrombocytopenia, anasarca, fever/hyperinflammatory status, organomegaly), renal dysfunction or characteristic bone marrow findings, and lymph node features consistent with iMCD. The definition was validated with an external cohort (the ACCELERATE Natural History Registry). The present international definition will facilitate a more precise and comprehensive approach to the diagnosis of iMCD-TAFRO.

International, evidence-based consensus treatment guidelines for idiopathic multicentric Castleman disease.

Castleman disease (CD) describes a group of heterogeneous hematologic disorders with characteristic histopathological features. CD can present with unicentric or multicentric (MCD) regions of lymph node enlargement. Some cases of MCD are caused by human herpesvirus-8 (HHV-8), whereas others are HHV-8-negative/idiopathic (iMCD). Treatment of iMCD is challenging, and outcomes can be poor because no uniform treatment guidelines exist, few systematic studies have been conducted, and no agreed upon response criteria have been described. The purpose of this paper is to establish consensus, evidence-based treatment guidelines based on the severity of iMCD to improve outcomes. An international Working Group of 42 experts from 10 countries was convened by the Castleman Disease Collaborative Network to establish consensus guidelines for the management of iMCD based on published literature, review of treatment effectiveness for 344 cases, and expert opinion. The anti-interleukin-6 monoclonal antibody siltuximab (or tocilizumab, if siltuximab is not available) with or without corticosteroids is the preferred first-line therapy for iMCD. In the most severe cases, adjuvant combination chemotherapy is recommended. Additional agents are recommended, tailored by disease severity, as second- and third-line therapies for treatment failures. Response criteria were formulated to facilitate the evaluation of treatment failure or success. These guidelines should help treating physicians to stratify patients based on disease severity in order to select the best available therapeutic option. An international registry for patients with CD (ACCELERATE, #NCT02817997) was established in October 2016 to collect patient outcomes to increase the evidence base for selection of therapies in the future.

International, evidence-based consensus diagnostic criteria for HHV-8-negative/idiopathic multicentric Castleman disease.

Human herpesvirus-8 (HHV-8)-negative, idiopathic multicentric Castleman disease (iMCD) is a rare and life-threatening disorder involving systemic inflammatory symptoms, polyclonal lymphoproliferation, cytopenias, and multiple organ system dysfunction caused by a cytokine storm often including interleukin-6. iMCD accounts for one third to one half of all cases of MCD and can occur in individuals of any age. Accurate diagnosis is challenging, because no standard diagnostic criteria or diagnostic biomarkers currently exist, and there is significant overlap with malignant, autoimmune, and infectious disorders. An international working group comprising 34 pediatric and adult pathology and clinical experts in iMCD and related disorders from 8 countries, including 2 physicians that are also iMCD patients, was convened to establish iMCD diagnostic criteria. The working group reviewed data from 244 cases, met twice, and refined criteria over 15 months (June 2015 to September 2016). The proposed consensus criteria require both Major Criteria (characteristic lymph node histopathology and multicentric lymphadenopathy), at least 2 of 11 Minor Criteria with at least 1 laboratory abnormality, and exclusion of infectious, malignant, and autoimmune disorders that can mimic iMCD. Characteristic histopathologic features may include a constellation of regressed or hyperplastic germinal centers, follicular dendritic cell prominence, hypervascularization, and polytypic plasmacytosis. Laboratory and clinical Minor Criteria include elevated C-reactive protein or erythrocyte sedimentation rate, anemia, thrombocytopenia or thrombocytosis, hypoalbuminemia, renal dysfunction or proteinuria, polyclonal hypergammaglobulinemia, constitutional symptoms, hepatosplenomegaly, effusions or edema, eruptive cherry hemangiomatosis or violaceous papules, and lymphocytic interstitial pneumonitis. iMCD consensus diagnostic criteria will facilitate consistent diagnosis, appropriate treatment, and collaborative research.

A benefit and the prospects of IL-6 inhibitors in idiopathic multicentric Castleman's disease.

Castleman's disease (CD), a heterogeneous group of lymphoproliferative disorders, is divided into unicentric CD (UCD) and multicentric CD (MCD) based on the number of regions of enlarged lymph nodes with characteristic histopathologic features. The clinical pictures and treatment differ greatly between these UCD and MCD. In MCD, cases of human herpesvirus 8-negative patients with unknown etiology are defined as idiopathic MCD (iMCD). Most cases of UCD are treatable by surgical excision. The prognosis of iMCD varies, and it may be challenging to achieve remission. Glucocorticoids are initiated as the first choice for therapy, but for glucocorticoid-resistant cases, interleukin (IL)-6 inhibition is initiated. However, an IL-6 inhibitor is not effective for all iMCD cases, and refractory cases occur despite these treatments. In this review, we briefly summarize the role of IL-6 in iMCD, and we discuss the efficacy that has been reported for tocilizumab (TCZ), the anti-IL-6 receptor antibody, for patients with iMCD in Japan. Factors predicting the therapeutic response to IL-6 remain to be identified, and the verification of the long-term safety of IL-6 inhibition is needed.

IL-6 inhibitor for the treatment of rheumatoid arthritis: A comprehensive review.

Tocilizumab (TCZ) is an interleukin-6 (IL-6) inhibitor used for the treatment of rheumatoid arthritis (RA). It was developed in 2008, and its effectiveness is supported by evidence from all over the world based on its first decade of use. Although the overall efficacy and safety profiles of TCZ are similar to those of tumor necrosis factor (TNF) inhibitors, TCZ displays certain differences. The most notable advantage of TCZ is its usefulness as a monotherapy. Additionally, TCZ is favorable in the improvement of systemic inflammatory symptoms such as anemia and fatigue. The low immunogenicity of TCZ contributes favorably to long-term drug retention. Due to frequent relapse after TCZ cessation, TCZ use should be tapered beyond remission. During TCZ therapy, C-reactive protein (CRP) is unable to recognize disease activity and the severity of infection. The most common adverse events (AEs) are infection and abnormalities in laboratory findings including dyslipidemia, neutropenia, thrombocytopenia, and abnormality of liver enzymes. TCZ obscures the symptoms of infection. Therefore, stealth infections without obvious CRP elevation can sometimes cause severe damage to patients. Lower intestinal perforation is an uncommon but serious AE in TCZ therapy. Further clinical investigations will continue to refine the IL-6 inhibitory strategy.

Tentative diagnostic criteria and disease severity classification for Castleman disease: A report of the research group on Castleman disease in Japan.

OBJECTIVES: To determine the tentative diagnostic criteria and disease severity classification for Castleman disease (CD) and describe the clinical and pathologic features among human herpesvirus 8 (HHV-8) negative idiopathic multicentric CD (iMCD) in the Japanese population. METHODS: We established the working groups for the research of CD in Japan and had meetings to discuss and define the tentative diagnostic criteria and disease severity classification for CD. We subsequently analyzed 142 patients classified into iMCD by using the nationwide Japanese patient registry. RESULTS: We proposed the preliminary diagnostic criteria and disease severity classification for CD based on our discussion. In addition, we made a proposal for the disease activity score. We identified clinical and pathological features of patients with iMCD diagnosed by these diagnostic criteria. In the disease severity classification, 37, 33 and 30% patients were categorized into mild, moderate and severe diseases, respectively. CONCLUSION: This is the first proposal for diagnosis and classification of CD by the Japanese group. Further studies are required to validate whether they can distinguish CD from other inflammatory diseases and to determine their sensitivity and specificity.

Prediction and experimental validation of a putative non-consensus binding site for transcription factor STAT3 in serum amyloid A gene promoter.

We previously demonstrated that though the human SAA1 gene shows no typical STAT3 response element (STAT3-RE) in its promoter region, STAT3 and the nuclear factor (NF-κB) p65 first form a complex following interleukin IL-1 and IL-6 (IL-1+6) stimulation, after which STAT3 interacts with a region downstream of the NF-κB RE in the SAA1 promoter. In this study, we employed a computational approach based on indirect read outs of protein-DNA contacts to identify a set of candidates for non-consensus STAT3 transcription factor binding sites (TFBSs). The binding of STAT3 to one of the predicted non-consensus TFBSs was experimentally confirmed through a dual luciferase assay and DNA affinity chromatography. The present study defines a novel STAT3 non-consensus TFBS at nt -75/-66 downstream of the NF-κB RE in the SAA1 promoter region that is required for NF-κB p65 and STAT3 to activate SAA1 transcription in human HepG2 liver cells. Our analysis builds upon the current understanding of STAT3 function, suggesting a wider array of mechanisms of STAT3 function in inflammatory response, and provides a useful framework for investigating novel TF-target associations with potential therapeutic implications.

Immunology and targeted therapy in Castleman disease.

INTRODUCTION: Castleman disease (CD) is a benign lymphoproliferative disease causing severe systemic inflammation. Interleukin-6 (IL-6) is a major pathogenesis of multicentric CD (MCD), but only 30-60% of patients respond to IL-6 inhibitors. Novel agents for IL-6 inhibitor-refractory cases are needed. Clinical data and samples are being collected on a large scale and the clinical, pathological, and pathogenetic aspects are being elucidated. AREAS COVERED: The pathological and clinical classification of CD is outlined. Focusing on idiopathic MCD (iMCD), this review identifies therapeutic targets and summarizes currently recommended drugs and promising therapeutic candidates. EXPERT OPINION: The pathogenesis of MCD has been implicated in the activation of the Janus kinase (JAK)-transcriptional signaling activator (STAT) 3 pathway and the phosphatidylinositol 3-kinase (PI3K)/Akt/mechanical target of rapamycin (mTOR) signaling pathway. iMCD-TAFRO (thrombocytopenia, anasarca, fever/elevated CRP, reticulin myelofibrosis/renal dysfunction, organ enlargement) is resistant to IL-6 inhibitors, and cyclosporine and mTOR inhibitors are sometimes effective. JAK inhibitors and mTOR inhibitors may be therapeutic agents for iMCD. Recently, we have shown that peripheral helper T (Tph) cell abnormalities are at the core of iMCD pathogenesis. Therapies targeting chemokine (C-X-C motif) ligand 13 (CXCL13) produced by Tph cells and blocking the Tph-CXCL13-B cell pathway may satisfy unmet need in refractory cases.

Predictive biomarkers of COVID-19 prognosis identified in Bangladesh patients and validated in Japanese cohorts.

Despite high vaccination rates globally, countries are still grappling with new COVID infections, and patients diagnosed as mild dying at home during outpatient treatment. Hence, this study aim to identify, then validate, biomarkers that could predict if newly infected COVID-19 patients would subsequently require hospitalization or could recover safely with medication as outpatients. Serum cytokine/chemokine data from 129 COVID-19 patients within 7 days after the onset of symptoms in Bangladesh were used as training data. The majority of patients were infected with the Omicron variant and over 88% were vaccinated. Patients were divided into those with mild symptoms who recovered, and those who deteriorated to moderate or severe illness. Using the Lasso method, 15 predictive markers were identified and used to classify patients into these two groups. The biomarkers were then validated in a cohort of 194 Covid patients in Japan with a predictive accuracy that exceeded 80% for patients infected with Delta and Omicron variants, and 70% for Wuhan and Alpha variants. In an environment of widespread vaccination, these biomarkers could help medical practitioners determine if newly infected COVID-19 patients will improve and can be managed on an out-patient basis, or if they will deteriorate and require hospitalization.

Peripheral helper-T-cell-derived CXCL13 is a crucial pathogenic factor in idiopathic multicentric Castleman disease.

Castleman disease (CD) is a rare lymphoproliferative disorder. Among subtypes of CD, idiopathic multicentric CD-not otherwise specified (iMCD-NOS) has a poor prognosis and its pathogenesis is largely unknown. Here we present a xenotransplantation model of iMCD-NOS pathogenesis. Immunodeficient mice, transplanted with lymph node (LN) cells from iMCD-NOS patients, develop iMCD-like lethal inflammation, while mice transplanted with LN cells from non-iMCD patients without inflammation serve as negative control. Grafts depleted of human CD3+ T cells fail to induce inflammation in vivo. Upon engraftment, peripheral helper T (Tph) cells expand and levels of human CXCL13 substantially increase in the sera of mice. A neutralizing antibody against human CXCL13 blocks development of inflammation and improves survival in the recipient mice. Our study thus indicates that Tph cells, producing CXCL13 play a critical role in the pathogenesis of iMCD-NOS, and establishes iMCD-NOS as an immunoregulatory disorder.

Down-regulation of hepcidin resulting from long-term treatment with an anti-IL-6 receptor antibody (tocilizumab) improves anemia of inflammation in multicentric Castleman disease.

Dysregulated production of hepcidin is implicated in anemia of inflammation, whereas interleukin-6 (IL-6) is a major inducer of hepcidin production. Overproduction of IL-6 is responsible for pathogenesis of multicentric Castleman disease (MCD), a rare lymphoproliferative disorder accompanied by systemic inflammatory responses and anemia. In this study, we investigated the roles of hepcidin and IL-6 in anemia of inflammation and the long-term effects of anti-IL-6 receptor antibody (tocilizumab) treatment on serum hepcidin and iron-related parameters in MCD patients. We found that tocilizumab treatment resulted in a rapid reduction of serum hepcidin-25 in 5 of 6 MCD patients. Long-term reductions, accompanied by progressive normalization of iron-related parameters and symptom improvement, were observed in 9 of 9 cases 1.5, 3, 6, and 12 months after the start of tocilizumab treatment. In in vitro experiments, IL-6-induced up-regulation of hepcidin mRNA in hepatoma cell lines was completely inhibited by tocilizumab but increased in the presence of patients' sera. Our results suggest that, although multiple factors affect serum hepcidin levels, IL-6 plays an essential role in the induction of hepcidin in MCD. This accounts for the long-term ameliorative effect of IL-6 blockage with tocilizumab on anemia by inhibiting hepcidin production in MCD patients.

A case of Behçet's disease treated with a humanized anti-interleukin-6 receptor antibody, tocilizumab.

A 47-year-old female patient with Behçet's disease had been treated with colchicine, prednisolone, cyclosporine A, and infliximab. Because she relapsed, however, treatment with tocilizumab, a humanized anti-interleukin 6 receptor antibody, was started. This treatment suppressed the patient's clinical manifestations, including ocular attacks, for 1 year and improved her visual acuity. This experience indicates that tocilizumab may constitute a therapeutic option for refractory Behçet's disease.

Anti-nucleocapsid antibodies enhance the production of IL-6 induced by SARS-CoV-2 N protein.

A cytokine storm induces acute respiratory distress syndrome, the main cause of death in coronavirus disease 2019 (COVID-19) patients. However, the detailed mechanisms of cytokine induction due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remain unclear. To examine the cytokine production in COVID-19, we mimicked the disease in SARS-CoV-2-infected alveoli by adding the lysate of SARS-CoV-2-infected cells to cultured macrophages or induced pluripotent stem cell-derived myeloid cells. The cells secreted interleukin (IL)-6 after the addition of SARS-CoV-2-infected cell lysate. Screening of 25 SARS-CoV-2 protein-expressing plasmids revealed that the N protein-coding plasmid alone induced IL-6 production. The addition of anti-N antibody further enhanced IL-6 production, but the F(ab')2 fragment did not. Sera from COVID-19 patients also enhanced IL-6 production, and sera from patients with severer disease induced higher levels of IL-6. These results suggest that anti-N antibody promotes IL-6 production in SARS-CoV-2-infected alveoli, leading to the cytokine storm of COVID-19.

Prompt Reduction in CRP, IL-6, IFN-γ, IP-10, and MCP-1 and a Relatively Low Basal Ratio of Ferritin/CRP Is Possibly Associated With the Efficacy of Tocilizumab Monotherapy in Severely to Critically Ill Patients With COVID-19.

Background and Aim: Tocilizumab, a humanized anti-IL-6 receptor antibody, has been used to treat severely to critically ill patients with COVID-19. A living systematic review with meta-analysis of recent RCTs indicates that the combination therapy of corticosteroids and tocilizumab produce better outcomes, while previous observational studies suggest that tocilizumab monotherapy is beneficial for substantial numbers of patients. However, what patients could respond to tocilizumab monotherapy remained unknown. Methods: In this retrospective study we evaluated the effects of tocilizumab monotherapy on the clinical characteristics, serum biomediator levels, viral elimination, and specific IgG antibody induction in 13 severely to critically ill patients and compared with those of dexamethasone monotherapy and dexamethasone plus tocilizumab. Results: A single tocilizumab administration led to a rapid improvement in clinical characteristics, inflammatory findings, and oxygen supply in 7 of 11 patients with severe COVID-19, and could recover from mechanical ventilation management (MVM) in 2 patients with critically ill COVID-19. Four patients exhibited rapidly worsening even after tocilizumab administration and required MVM and additional methylprednisolone treatment. Tocilizumab did not delay viral elimination or inhibit IgG production specific for the virus, whereas dexamethasone inhibited IgG induction. A multiplex cytokine array system revealed a significant increase in the serum expression of 54 out of 80 biomediators in patients with COVID-19 compared with that in healthy controls. Compared with those who promptly recovered in response to tocilizumab, patients requiring MVM showed a significantly higher ratio of basal level of ferritin/CRP and a persistent increase in the levels of CRP and specific cytokines and chemokines including IL-6, IFN-γ, IP-10, and MCP-1. The basal high ratio of ferritin/CRP was also associated with clinical deterioration even in patients treated with dexamethasone and tocilizumab. Conclusion: Tocilizumab as monotherapy has substantial beneficial effects in some patients with severe COVID-19, who showed a relatively low level of the ratio of ferritin/CRP and prompt reduction in CRP, IL-6, IFN-γ, IP-10, and MCP-1. The high ratio of ferritin/CRP is associated with rapid worsening of pneumonia. Further evaluation is warranted to clarify whether tocilizumab monotherapy or its combination with corticosteroid is preferred for severely to critically ill patients with COVID-19.

International evidence-based consensus diagnostic and treatment guidelines for unicentric Castleman disease.

Castleman disease (CD) includes a group of rare and heterogeneous disorders with characteristic lymph node histopathological abnormalities. CD can occur in a single lymph node station, which is referred to as unicentric CD (UCD). CD can also involve multicentric lymphadenopathy and inflammatory symptoms (multicentric CD [MCD]). MCD includes human herpesvirus-8 (HHV-8)-associated MCD, POEMS-associated MCD, and HHV-8-/idiopathic MCD (iMCD). The first-ever diagnostic and treatment guidelines were recently developed for iMCD by an international expert consortium convened by the Castleman Disease Collaborative Network (CDCN). The focus of this report is to establish similar guidelines for the management of UCD. To this purpose, an international working group of 42 experts from 10 countries was convened to establish consensus recommendations based on review of treatment in published cases of UCD, the CDCN ACCELERATE registry, and expert opinion. Complete surgical resection is often curative and is therefore the preferred first-line therapy, if possible. The management of unresectable UCD is more challenging. Existing evidence supports that asymptomatic unresectable UCD may be observed. The anti-interleukin-6 monoclonal antibody siltuximab should be considered for unresectable UCD patients with an inflammatory syndrome. Unresectable UCD that is symptomatic as a result of compression of vital neighboring structures may be rendered amenable to resection by medical therapy (eg, rituximab, steroids), radiotherapy, or embolization. Further research is needed in UCD patients with persisting constitutional symptoms despite complete excision and normal laboratory markers. We hope that these guidelines will improve outcomes in UCD and help treating physicians decide the best therapeutic approach for their patients.

[New therapeutic strategy for autoimmune and chronic inflammatory disease based on clinical results using IL-6 blocking therapy with a humanized anti-IL-6 receptor antibody].

Remarkable clinical effects were observed by IL-6 blockage with a humanized anti IL-6 receptor antibody in patients with Castleman's disease, rheumatoid arthritis, and juvenile inflammatory arthritis. This evidence suggests that the hyper-function of IL-6 is an essential key cytokine in the pathogenesis of the above diseases, in which many cytokines, chemokines, and inflammatory molecules are activated. We found, for example, TNF-alpha blocking therapy showed a reduction of acute phase proteins, such as CRP and SAA, however, the IL-6 blockade induced not only reduction but also normalization of CRP and SAA serum levels. To elucidate this in vivo phenomenon, we analyzed the expression of cytokine inducing CRP and SAA mRNA with the intracellular signal transduction mechanism in vitro. The results, indicated that the IL-6 signal was essential though the activation of STAT3 for the induction and augmentation of CRP or SAA by the associated stimulation with TNF-alpha or IL-1. Recently, it is now known that IL-6 is a regulatory molecule in the induction of Th17 cells with TGF-beta. Therefore, IL-6 blockage may potentially improve autoimmune diseases, beginning with the pathogenic initiation phase. We believe that unknown pathogenic inflammatory phenomena can be clarified using this analytical strategy and cytokine blocking therapy. Furthermore, in the future we hope to induce complete remission of autoimmune diseases by using cytokine blockage freely.

The IL-6R alpha chain controls lung CD4+CD25+ Treg development and function during allergic airway inflammation in vivo.

The cytokine IL-6 acts via a specific receptor complex that consists of the membrane-bound IL-6 receptor (mIL-6R) or the soluble IL-6 receptor (sIL-6R) and glycoprotein 130 (gp130). In this study, we investigated the role of IL-6R components in asthma. We observed increased levels of sIL-6R in the airways of patients with allergic asthma as compared to those in controls. In addition, local blockade of the sIL-6R in a murine model of late-phase asthma after OVA sensitization by gp130-fraction constant led to suppression of Th2 cells in the lung. By contrast, blockade of mIL-6R induced local expansion of Foxp3-positive CD4+CD25+ Tregs with increased immunosuppressive capacities. CD4+CD25+ but not CD4+CD25- lung T cells selectively expressed the IL-6R alpha chain and showed IL-6-dependent STAT-3 phosphorylation. Finally, in an in vivo transfer model of asthma in immunodeficient Rag1 mice, CD4+CD25+ T cells isolated from anti-IL-6R antibody-treated mice exhibited marked immunosuppressive and antiinflammatory functions. IL-6 signaling therefore controls the balance between effector cells and Tregs in the lung by means of different receptor components. Furthermore, inhibition of IL-6 signaling emerges as a novel molecular approach for the treatment of allergic asthma.

The Role of Interleukin-6 in Castleman Disease.

Since its discovery, improvements in treating Castleman disease and its variants have centered on interleukin-6 (IL-6). IL-6 was discovered from T-cell factors (BCDF or BSF-2), which induced B-cell maturation. Most symptoms of the plasma cell variant of Castleman disease are linked to the hyperfunction of IL-6, constitutively produced in the affected lymph nodes (1989), suggesting IL-6 is key in the pathogenesis of multicentric Castleman disease (MCD). The results of several studies have shown that most MCD symptoms and abnormal laboratory results are improved by anti-IL-6 MCD treatments, such as tocilizumab, a humanized anti-IL-6 receptor antibody, and siltuximab, an anti-IL-6 antibody.

Anti-interleukin 6 receptor antibody tocilizumab reduces the level of serum hepcidin in patients with multicentric Castleman's disease.

We report two cases of multicentric Castleman's disease (MCD) whose serum hepcidin levels were rapidly down-regulated by administration of tocilizumab, an anti- interleukin 6 (IL-6) receptor antibody. Our results indicate that IL-6-induced hepcidin over-production may be involved in the pathophysiology of microcytic anemia commonly observed in this disease.