JNK2-MMP-9 axis facilitates the progression of intracranial aneurysms.

Intracranial aneurysm (IA) can cause subarachnoid hemorrhage or some other hemorrhagic stroke after rupture. Because of the poor outcome in spite of the intensive medical care after the onset of hemorrhage, the development of a novel therapeutic strategy like medical therapy to prevent the progression of the disease becomes a social need. As the reduction of arterial stiffness due to the degeneration of the extracellular matrix via Matrix Metalloproteinases (MMPs) becomes one of the central machineries leading to the progression of IAs through a series of studies, factors regulating the expression or the activity of MMPs could be a therapeutic target. In the present study, specimens from human IA lesions and the animal model of IAs were used to examine the expression of c-Jun N-terminal kinase (JNK) which might exacerbate expressions of MMPs in the lesions to weaken arterial walls resulting in the progression of the disease. In some human IA lesions examined, the expression of p-JNK, the activated form of JNK, could be detected mostly in the medial smooth muscle cells. In IA lesions induced in rats, the activation of JNK was induced during the progression of the disease and accompanied with the activation of downstream transcriptional factor c-Jun and importantly with the expression of MMP-2 or -9. The genetic deletion of Jnk2, not Jnk1, in mice significantly prevented the incidence of IAs with the suppression of the expression of MMP-2 or MMP-9. These results combined together have suggested the crucial role of JNK in the progression of IAs through regulating the expression of MMPs. The results from the present study provides the novel insights about the pathogenesis of IA progression and also about the therapeutic target.

The potential of disulfiram as a drug to improve the prognosis after the onset of subarachnoid hemorrhage.

Subarachnoid hemorrhage due to rupture of intracranial aneurysms has a poor outcome, making this disease being the social problem. Inflammation evoked by the increase in intracranial pressure and the clot in the subarachnoid space after the onset of SAH exacerbates neuronal death and vasospasm, resulting in the poor outcome and severe aftereffects. Here, FROUNT mediates CCR2 and CCR5 signaling as an intracellular molecule binding to these chemoattractant receptors which facilitate the migration of inflammatory cells, such as macrophages, in situ to trigger inflammation there. Animal model of subarachnoid hemorrhage was established in rats through intrathecal injection of autologous blood. The effect of the FROUNT inhibitor, disulfiram, on survival rate, neuronal death in hippocampus or vasospasm was then examined. The intrathecal administration of disulfiram significantly suppressed the infiltration of CD68-positive macrophages and myeloperoxidase-positive neutrophils toward the clot in the cistern in situ. In this condition, disulfiram ameliorated the death of animals after the onset of subarachnoid hemorrhage in rats. In addition, disulfiram suppressed both the two major events after subarachnoid hemorrhage, the neuronal death in hippocampus and vasospasm. The pharmacological inhibition of CCR2 and CCR5 signaling by disulfiram could thus be the therapeutic strategy to improve the outcome of subarachnoid hemorrhage.

Integrated single cell analysis reveals co-evolution of malignant B cells and tumor micro-environment in transformed follicular lymphoma.

Histological transformation of follicular lymphoma (FL) to aggressive forms is associated with poor outcome. Phenotypic consequences of this evolution and its impact on the tumor microenvironment (TME) remain unknown. We perform single-cell whole genome sequencing (scWGS) and transcriptome sequencing (scWTS) of 11 paired pre/post-transformation patient samples and scWTS of additional samples from patients without transformation. Our analysis reveals evolutionary dynamics of transformation at single-cell resolution, highlighting a shifting TME landscape, with an emerging immune-cell exhaustion signature, co-evolving with the shifting malignant B phenotype in a regulatory ecosystem. Integration of scWGS and scWTS identifies malignant cell pathways upregulated during clonal tumor evolution. Using multi-color immunofluorescence, we transfer these findings to a TME-based transformation biomarker, subsequently validated in two independent pretreatment cohorts. Taken together, our results provide a comprehensive view of the combined genomic and phenotypic evolution of malignant cells during transformation and shifting crosstalk between malignant cells and the TME.

TRAF3 loss-of-function reveals the noncanonical NF-κB pathway as a therapeutic target in diffuse large B cell lymphoma.

Here, we report recurrent focal deletions of the chr14q32.31-32 locus, including TRAF3, a negative regulator of NF-κB signaling, in de novo diffuse large B cell lymphoma (DLBCL) (24/324 cases). Integrative analysis revealed an association between TRAF3 copy number loss with accumulation of NIK, the central noncanonical (NC) NF-κB kinase, and increased NC NF-κB pathway activity. Accordingly, TRAF3 genetic ablation in isogenic DLBCL model systems caused upregulation of NIK and enhanced NC NF-κB downstream signaling. Knockdown or pharmacological inhibition of NIK in TRAF3-deficient cells differentially impaired their proliferation and survival, suggesting an acquired onco-addiction to NC NF-κB. TRAF3 ablation also led to exacerbated secretion of the immunosuppressive cytokine IL-10. Coculturing of TRAF3-deficient DLBCL cells with CD8+ T cells impaired the induction of Granzyme B and interferon (IFN) γ, which were restored following neutralization of IL-10. Our findings corroborate a direct relationship between TRAF3 genetic alterations and NC NF-κB activation, and highlight NIK as a potential therapeutic target in a defined subset of DLBCL.

Correction: Verification in the Early Stages of the COVID-19 Pandemic: Sentiment Analysis of Japanese Twitter Users.

[This corrects the article DOI: 10.2196/37881.].

C5a-C5AR1 axis as a potential trigger of the rupture of intracranial aneurysms.

Recent studies have indicated the involvement of neutrophil-mediated inflammatory responses in the process leading to intracranial aneurysm (IA) rupture. Receptors mediating neutrophil recruitment could thus be therapeutic targets of unruptured IAs. In this study, complement C5a receptor 1 (C5AR1) was picked up as a candidate that may cause neutrophil-dependent inflammation in IA lesions from comprehensive gene expression profile data acquired from rat and human samples. The induction of C5AR1 in IA lesions was confirmed by immunohistochemistry; the up-regulations of C5AR1/C5ar1 stemmed from infiltrated neutrophils, which physiologically express C5AR1/C5ar1, and adventitial fibroblasts that induce C5AR1/C5ar1 in human/rat IA lesions. In in vitro experiments using NIH/3T3, a mouse fibroblast-like cell line, induction of C5ar1 was demonstrated by starvation or pharmacological inhibition of mTOR signaling by Torin1. Immunohistochemistry and an experiment in a cell-free system using recombinant C5 protein and recombinant Plasmin indicated that the ligand of C5AR1, C5a, could be produced through the enzymatic digestion by Plasmin in IA lesions. In conclusion, we have identified a potential contribution of the C5a-C5AR1 axis to neutrophil infiltration as well as inflammatory responses in inflammatory cells and fibroblasts of IA lesions. This cascade may become a therapeutic target to prevent the rupture of IAs.

Time-of-flight and black-blood MRI to study intracranial arteries in rats.

Intracranial aneurysms (IAs) are usually incidentally discovered by magnetic resonance imaging (MRI). Once discovered, the risk associated with their treatment must be balanced with the risk of an unexpected rupture. Although clinical observations suggest that the detection of contrast agent in the aneurysm wall using a double-inversion recovery black-blood (BB) sequence may point to IA wall instability, the exact meaning of this observation is not understood. Validation of reliable diagnostic markers of IA (in)stability is of utmost importance to deciding whether to treat or not an IA. To longitudinally investigate IA progression and enhance our understanding of this devastating disease, animal models are of great help. The aim of our study was to improve a three-dimensional (3D)-time-of-flight (TOF) sequence and to develop a BB sequence on a standard preclinical 3-T MRI unit to investigate intracranial arterial diseases in rats. We showed that our 3D-TOF sequence allows reliable measurements of intracranial artery diameters, inter-artery distances, and angles between arteries and that our BB sequence enables us to visualize intracranial arteries. We report the first BB-MRI sequence to visualize intracranial arteries in rats using a preclinical 3-T MRI unit. This sequence could be useful for a large community of researchers working on intracranial arterial diseases.Relevance statement We developed a black-blood MRI sequence to study vessel wall enhancement in rats with possible application to understanding IAs instability and finding reliable markers for clinical decision-making.Key points• Reliable markers of aneurysm stability are needed for clinical decision.• Detection of contrast enhancement in the aneurysm wall may be associated with instability.• We developed a black-blood MRI sequence in rats to be used to study vessel wall enhancement of IAs.

Application of deep neural survival networks to the development of risk prediction models for diabetes mellitus, hypertension, and dyslipidemia.

OBJECTIVES: : Although numerous risk prediction models have been proposed, few such models have been developed using neural network-based survival analysis. We developed risk prediction models for three cardiovascular disease risk factors (diabetes mellitus, hypertension, and dyslipidemia) among a working-age population in Japan using DeepSurv, a deep feed-forward neural network. METHODS: : Data were obtained from the Japan Epidemiology Collaboration on Occupational Health Study. A total of 51 258, 44 197, and 31 452 individuals were included in the development of risk models for diabetes mellitus, hypertension, and dyslipidemia, respectively; two-thirds of whom were used to develop prediction models, and the rest were used to validate the models. We compared the performances of DeepSurv-based models with those of prediction models based on the Cox proportional hazards model. RESULTS: : The area under the receiver-operating characteristic curve was 0.878 [95% confidence interval (CI) = 0.864-0.892] for diabetes mellitus, 0.835 (95% CI = 0.826-0.845) for hypertension, and 0.826 (95% CI = 0.817-0.835) for dyslipidemia. Compared with the Cox proportional hazards-based models, the DeepSurv-based models had better reclassification performance [diabetes mellitus: net reclassification improvement (NRI) = 0.474, P  ≤ 0.001; hypertension: NRI = 0.194, P  ≤ 0.001; dyslipidemia: NRI = 0.397, P  ≤ 0.001] and discrimination performance [diabetes mellitus: integrated discrimination improvement (IDI) = 0.013, P  ≤ 0.001; hypertension: IDI = 0.007, P  ≤ 0.001; and dyslipidemia: IDI = 0.043, P  ≤ 0.001]. CONCLUSION: : This study suggests that DeepSurv has the potential to improve the performance of risk prediction models for cardiovascular disease risk factors.

Spatially Resolved Tumor Microenvironment Predicts Treatment Outcomes in Relapsed/Refractory Hodgkin Lymphoma.

PURPOSE: About a third of patients with relapsed or refractory classic Hodgkin lymphoma (r/r CHL) succumb to their disease after high-dose chemotherapy followed by autologous stem-cell transplantation (HDC/ASCT). Here, we aimed to describe spatially resolved tumor microenvironment (TME) ecosystems to establish novel biomarkers associated with treatment failure in r/r CHL. PATIENTS AND METHODS: We performed imaging mass cytometry (IMC) on 71 paired primary diagnostic and relapse biopsies using a marker panel specific to CHL biology. For each cell type in the TME, we calculated a spatial score measuring the distance of nearest neighbor cells to the malignant Hodgkin Reed Sternberg cells within the close interaction range. Spatial scores were used as features in prognostic model development for post-ASCT outcomes. RESULTS: Highly multiplexed IMC data revealed shared TME patterns in paired diagnostic and early r/r CHL samples, whereas TME patterns were more divergent in pairs of diagnostic and late relapse samples. Integrated analysis of IMC and single-cell RNA sequencing data identified unique architecture defined by CXCR5+ Hodgkin and Reed Sternberg (HRS) cells and their strong spatial relationship with CXCL13+ macrophages in the TME. We developed a prognostic assay (RHL4S) using four spatially resolved parameters, CXCR5+ HRS cells, PD1+CD4+ T cells, CD68+ tumor-associated macrophages, and CXCR5+ B cells, which effectively separated patients into high-risk versus low-risk groups with significantly different post-ASCT outcomes. The RHL4S assay was validated in an independent r/r CHL cohort using a multicolor immunofluorescence assay. CONCLUSION: We identified the interaction of CXCR5+ HRS cells with ligand-expressing CXCL13+ macrophages as a prominent crosstalk axis in relapsed CHL. Harnessing this TME biology, we developed a novel prognostic model applicable to r/r CHL biopsies, RHL4S, opening new avenues for spatial biomarker development.

Verification in the Early Stages of the COVID-19 Pandemic: Sentiment Analysis of Japanese Twitter Users.

BACKGROUND: The COVID-19 pandemic prompted global behavioral restrictions, impacting public mental health. Sentiment analysis, a tool for assessing individual and public emotions from text data, gained importance amid the pandemic. This study focuses on Japan's early public health interventions during COVID-19, utilizing sentiment analysis in infodemiology to gauge public sentiment on social media regarding these interventions. OBJECTIVE: This study aims to investigate shifts in public emotions and sentiments before and after the first state of emergency was declared in Japan. By analyzing both user-generated tweets and retweets, we aim to discern patterns in emotional responses during this critical period. METHODS: We conducted a day-by-day analysis of Twitter (now known as X) data using 4,894,009 tweets containing the keywords "corona," "COVID-19," and "new pneumonia" from March 23 to April 21, 2020, approximately 2 weeks before and after the first declaration of a state of emergency in Japan. We also processed tweet data into vectors for each word, employing the Fuzzy-C-Means (FCM) method, a type of cluster analysis, for the words in the sentiment dictionary. We set up 7 sentiment clusters (negative: anger, sadness, surprise, disgust; neutral: anxiety; positive: trust and joy) and conducted sentiment analysis of the tweet groups and retweet groups. RESULTS: The analysis revealed a mix of positive and negative sentiments, with "joy" significantly increasing in the retweet group after the state of emergency declaration. Negative emotions, such as "worry" and "disgust," were prevalent in both tweet and retweet groups. Furthermore, the retweet group had a tendency to share more negative content compared to the tweet group. CONCLUSIONS: This study conducted sentiment analysis of Japanese tweets and retweets to explore public sentiments during the early stages of COVID-19 in Japan, spanning 2 weeks before and after the first state of emergency declaration. The analysis revealed a mix of positive (joy) and negative (anxiety, disgust) emotions. Notably, joy increased in the retweet group after the emergency declaration, but this group also tended to share more negative content than the tweet group. This study suggests that the state of emergency heightened positive sentiments due to expectations for infection prevention measures, yet negative information also gained traction. The findings propose the potential for further exploration through network analysis.

Scientific techniques in adolescent and young adult classic Hodgkin lymphoma.

Understanding the tumor microenvironment and genomic landscape is crucial for better prediction of treatment outcomes and developing novel therapies in Hodgkin lymphoma (HL). Recent advancements in genomics have enabled researchers to gain deeper insights into the genomic characteristics of HL at both single-cell resolution and the whole genome level. The use of noninvasive methods such as liquid biopsies and formalin-fixed paraffin-embedded-based imaging techniques has expanded the possibilities of applying cutting-edge analyses to routine clinically available samples. Collaborative efforts between adult and pediatric group are imperative to translate novel findings into routine patient care.

[Inflammatory Responses in Hemorrhagic Stroke].

Inflammation is triggered by various intrinsic and extrinsic stimuli as a protective machinery to maintain homeostasis in the human body. Usually, it is magnified in intensity initially and regresses rapidly afterwards; this phenomenon is called acute inflammation. However, it occasionally lasts a long time; this phenomenon is called chronic inflammation. Induction of some specific machineries, i.e., formation of a positive feedback loop, inflammatory cell infiltration, and changes in tissue architecture, is required for the transition to chronic inflammation; this differentiates chronic and acute inflammation in nature. Chronic inflammation is a common pathogenesis of various diseases, including cancer, vascular disease, and stroke. Recent experimental studies have clarified the crucial role of inflammatory responses in the development and progression of hemorrhagic stroke mediated by tissue destruction or some other aspects of diseases. In this review, we summarize the research findings of the role of inflammation in hemorrhagic stroke.

Clinicopathological characteristics associated with the engraftment of patient lymphoma cells in NOG mice.

Patient-derived xenograft (PDX) mouse models are useful for deepening our understanding of the biology of malignant lymphoma; however, factors associated with the success of the PDX lymphoma model are largely unknown. We retrospectively analyzed the characteristics of 66 xenotransplantations from 65 patients. In all, 43 (65%) specimens were obtained from patients aged > 60 years, and 42 (64%) specimens were obtained at diagnosis. Specimens were obtained from patients with the following diseases: diffuse large B-cell lymphoma (n = 30), intravascular large B-cell lymphoma (n = 12), follicular lymphoma (n = 8), peripheral T-cell lymphoma (n = 7), mantle cell lymphoma (n = 2), and other (n = 7). The specimens were sourced mainly from bone marrow (n = 31, 47%) and extranodal tumors (n = 13, 20%). Engraftment was successful in 33/66 (50%) xenotransplantations. The median age of patients who provided successful specimens was significantly higher than that for unsuccessful specimens (p = 0.013). Specimens with a high proportion of tumor cells in the graft and those obtained from patients with relapsed/refractory disease showed higher tendencies toward successful engraftment. Taken together, these data suggest that tumor cells with a highly malignant potential might have a high likelihood of engraftment.

Hypoxic microenvironment as a crucial factor triggering events leading to rupture of intracranial aneurysm.

Subarachnoid hemorrhage being the rupture of intracranial aneurysm (IA) as a major cause has quite poor prognosis, despite the modern technical advances. Thereby, the mechanisms underlying the rupture of lesions should be clarified. Recently, we and others have clarified the formation of vasa vasorum in IA lesions presumably for inflammatory cells to infiltrate in lesions as the potential histopathological alternation leading to rupture. In the present study, we clarified the origin of vasa vasorum as arteries located at the brain surface using 3D-immunohistochemistry with tissue transparency. Using Hypoxyprobe, we then found the presence of hypoxic microenvironment mainly at the adventitia of intracranial arteries where IA is formed. In addition, the production of vascular endothelial growth factor (VEGF) from cultured macrophages in such a hypoxic condition was identified. Furthermore, we found the accumulation of VEGF both in rupture-prone IA lesions induced in a rat model and human unruptured IA lesions. Finally, the VEGF-dependent induction of neovessels from arteries on brain surface was confirmed. The findings from the present study have revealed the potential role of hypoxic microenvironment and hypoxia-induced VEGF production as a machinery triggering rupture of IAs via providing root for inflammatory cells in lesions to exacerbate inflammation.

Novel insights into Hodgkin lymphoma biology by single-cell analysis.

The emergence and rapid development of single-cell technologies mark a paradigm shift in cancer research. Various technology implementations represent powerful tools to understand cellular heterogeneity, identify minor cell populations that were previously hard to detect and define, and make inferences about cell-to-cell interactions at single-cell resolution. Applied to lymphoma, recent advances in single-cell RNA sequencing have broadened opportunities to delineate previously underappreciated heterogeneity of malignant cell differentiation states and presumed cell of origin, and to describe the composition and cellular subsets in the ecosystem of the tumor microenvironment (TME). Clinical deployment of an expanding armamentarium of immunotherapy options that rely on targets and immune cell interactions in the TME emphasizes the requirement for a deeper understanding of immune biology in lymphoma. In particular, classic Hodgkin lymphoma (CHL) can serve as a study paradigm because of its unique TME, featuring infrequent tumor cells among numerous nonmalignant immune cells with significant interpatient and intrapatient variability. Synergistic to advances in single-cell sequencing, multiplexed imaging techniques have added a new dimension to describing cellular cross talk in various lymphoma entities. Here, we comprehensively review recent progress using novel single-cell technologies with an emphasis on the TME biology of CHL as an application field. The described technologies, which are applicable to peripheral blood, fresh tissues, and formalin-fixed samples, hold the promise to accelerate biomarker discovery for novel immunotherapeutic approaches and to serve as future assay platforms for biomarker-informed treatment selection, including immunotherapies.

Associations Between Drug Treatments and the Risk of Aneurysmal Subarachnoid Hemorrhage: a Systematic Review and Meta-analysis.

There is increasing interest in drug therapy for preventing aneurysmal subarachnoid hemorrhage (aSAH). We aimed to comprehensively evaluate the association between drug use and the risk of aSAH. We searched PubMed and Scopus from the databases' inception until December 2021. Observational studies reporting the association between any drug therapy and aSAH were included. The odds ratios (ORs) for each drug used in aSAH were meta-analyzed with a random-effect model. According to the systematic review, 25 observational studies were eligible for the present study. Four therapeutic purpose-based classes (e.g., lipid-lowering agents) and 14 mechanism-based classes (e.g., statins) were meta-analyzed. Anti-hypertensive agents (OR, 0.50; 95% confidence interval [95% CI], 0.33-0.74), statins (OR, 0.55; 95% CI, 0.35-0.85), biguanides (OR, 0.57; 95% CI, 0.34-0.96), and acetylsalicylic acid (ASA) (OR, 0.62; 95% CI, 0.41-0.94) were inversely associated with the risk of aSAH. Non-ASA non-steroidal anti-inflammatory drugs (OR, 1.73; 95% CI, 1.07-2.79), selective cyclooxygenase-2 inhibitors (OR, 2.04; 95% CI, 1.24-3.35), vitamin K antagonists (OR, 1.50; 95% CI, 1.18-1.91), and dipyridamole (OR, 1.77; 95% CI, 1.23-2.54) were positively associated with the incidence of aSAH. There was also a trend toward a positive association between glucocorticoids (OR, 1.38; 95% CI, 0.97-1.94) and aSAH. The present study suggests that anti-hypertensive agents, statins, biguanides, and ASA are candidate drugs for preventing aSAH. By contrast, several drugs (e.g., anti-thrombotic drugs) may increase the risk of aSAH. Thus, the indications of these drugs in patients with intracranial aneurysms should be carefully determined.

Identification of The Unique Subtype of Macrophages in Aneurysm Lesions at the Growth Phase.

OBJECTIVES: Recent experimental studies have defined intracranial aneurysms as a macrophage-mediated chronic inflammatory disease affecting intracranial arteries. Although there are various subtypes in macrophages, what type of macrophages is present in lesions during the disease development remains to be elucidated. METHODS: The previously-established aneurysm model of rats was used. Macrophages were labeled with the fluorescent protein and isolated by a laser-microdissection method. The comprehensive gene expression profile analyses and gene ontology analyses was then done to identify a macrophage subtype present in lesions at the growth phase. RESULTS: The gene expression profile data of total 52 macrophages infiltrating into the lesions was acquired. The principal component analysis revealed the monotonous macrophage subtype. By comparing the profile identified with one from in vitro-differentiated M0 or M1 macrophages, the macrophages in the lesions were belonged to the simple and unique subtype. Because the perception of signaling from nervous system was highlighted as up-represented terms through gene ontology analyses, the macrophage subtype in lesions at the growth phase might be differentiated under the influence of nervous system in the microenvironment. The histopathological examinations supported the above notion by confirming the presence of nerves in the adventitia. CONCLUSIONS: The findings from the present study have provided the useful insights about the macrophage subtype in aneurysm lesions at the growth phase and also proposed its ability as a therapeutic target.

Single-cell profiling reveals a memory B cell-like subtype of follicular lymphoma with increased transformation risk.

Follicular lymphoma (FL) is an indolent cancer of mature B-cells but with ongoing risk of transformation to more aggressive histology over time. Recurrent mutations associated with transformation have been identified; however, prognostic features that can be discerned at diagnosis could be clinically useful. We present here comprehensive profiling of both tumor and immune compartments in 155 diagnostic FL biopsies at single-cell resolution by mass cytometry. This revealed a diversity of phenotypes but included two recurrent patterns, one which closely resembles germinal center B-cells (GCB) and another which appears more related to memory B-cells (MB). GCB-type tumors are enriched for EZH2, TNFRSF14, and MEF2B mutations, while MB-type tumors contain increased follicular helper T-cells. MB-type and intratumoral phenotypic diversity are independently associated with increased risk of transformation, supporting biological relevance of these features. Notably, a reduced 26-marker panel retains sufficient information to allow phenotypic profiling of future cohorts by conventional flow cytometry.

Endothelial cell malfunction in unruptured intracranial aneurysm lesions revealed using a 3D-casted mold.

Intracranial aneurysms (IA) are major causes of devastating subarachnoid hemorrhages. They are characterized by a chronic inflammatory process in the intracranial arterial walls triggered and modified by hemodynamic force loading. Because IA lesion morphology is complex, the blood flow conditions loaded on endothelial cells in each portion of the lesion in situ vary greatly. We created a 3D-casted mold of the human unruptured IA lesion and cultured endothelial cells on this model; it was then perfused with culture media to model physiological flow conditions. Gene expression profiles of endothelial cells in each part of the IA lesion were then analyzed. Comprehensive gene expression profile analysis revealed similar gene expression patterns in endothelial cells from each part of the IA lesion but gene ontology analysis revealed endothelial cell malfunction within the IA lesion. Histopathological examination, electron microscopy, and immunohistochemical analysis indicated that endothelial cells within IA lesions are damaged and dysfunctional. Thus, our findings reveal endothelial cell malfunction in IA lesions and provided new insights into IA pathogenesis.