Ensuring Transfusion Safety: Screening Blood Donors for Human Parvovirus B19.

Ensuring the safety of blood and blood products is a vital aspect of healthcare. The potential for transmission of pathogens through blood and blood products makes transfusion safety a significant concern. Despite advancements in testing methodologies, donated blood products still pose a risk for infection transmission. Human parvovirus B19 (B19V) is a small, single-stranded, non-enveloped DNA virus transmissible parenterally by blood transfusion. B19V causes a wide range of clinical manifestations, which is generally harmless in healthy individuals. B19V infection may cause severe complications, such as aplastic crises, as it affects erythrocyte progenitor cells in individuals with increased erythrocyte turnover. Additionally, B19V can be transmitted from pregnant women to their foetus, potentially causing hydrops fetalis and foetal death. The potential for transmission through blood and blood products makes B19V a significant concern for transfusion safety. In response to the growing recognition of B19V's impact on transfusion safety, various international health organisations have introduced guidelines to minimise its transmission through blood and plasma products. However, the implementation of these guidelines varies globally, with some regions, such as India, still lacking formal protocols for B19V monitoring. This review article explores the existing methodologies for screening blood donors for B19V, assesses the associated transfusion risks, and considers the implications for public health and clinical practice. By emphasising advancements in diagnostic techniques and the challenges of their implementation, this article provides a comprehensive overview of efforts to reduce the transmission of B19V through blood transfusions, thereby ensuring safer blood supplies and improved patient outcomes.

Increased parvovirus B19 seropositivity in healthy blood donors in India.

A core component of every blood program is the supply of safe blood and blood products. The elevated risk of transmission through these products is due to parvovirus B19 (B19V) resistance to the virus inactivation procedures. Our study aimed to screen asymptomatic blood donors for B19V at a tertiary care hospital in Chennai, Tamil Nadu, between September 2020 and June 2021. Sera from 106 healthy blood donors who tested negative for Human immunodeficiency virus (HIV), Hepatitis B surface antigen (HBsAg), Hepatitis C virus (HCV), syphilis, and malaria were tested for anti-B19V IgM and IgG using a qualitative indirect enzyme-linked immunosorbent assay (ELISA). In the study population, 23.5% (n = 25) of donors tested IgM positive, 38.6% (n = 41) tested IgG positive, and 7.5% (n = 8) tested positive for both IgM and IgG. A proportion of 61.3% (n = 65) of the blood donors tested IgG negative, suggesting they had no past B19V infection. B19V DNA was not detected in any of the subjects. The high seroprevalence of IgM indicates that blood donors may have been recently exposed to B19V, potentially posing a risk to immunocompromised individuals and those with hematological stress. Further longitudinal studies with a larger sample size are recommended to better understand the risk of B19V transfusion transmission.

Fibrin drives thromboinflammation and neuropathology in COVID-19.

Life-threatening thrombotic events and neurological symptoms are prevalent in COVID-19 and are persistent in patients with long COVID experiencing post-acute sequelae of SARS-CoV-2 infection1-4. Despite the clinical evidence1,5-7, the underlying mechanisms of coagulopathy in COVID-19 and its consequences in inflammation and neuropathology remain poorly understood and treatment options are insufficient. Fibrinogen, the central structural component of blood clots, is abundantly deposited in the lungs and brains of patients with COVID-19, correlates with disease severity and is a predictive biomarker for post-COVID-19 cognitive deficits1,5,8-10. Here we show that fibrin binds to the SARS-CoV-2 spike protein, forming proinflammatory blood clots that drive systemic thromboinflammation and neuropathology in COVID-19. Fibrin, acting through its inflammatory domain, is required for oxidative stress and macrophage activation in the lungs, whereas it suppresses natural killer cells, after SARS-CoV-2 infection. Fibrin promotes neuroinflammation and neuronal loss after infection, as well as innate immune activation in the brain and lungs independently of active infection. A monoclonal antibody targeting the inflammatory fibrin domain provides protection from microglial activation and neuronal injury, as well as from thromboinflammation in the lung after infection. Thus, fibrin drives inflammation and neuropathology in SARS-CoV-2 infection, and fibrin-targeting immunotherapy may represent a therapeutic intervention for patients with acute COVID-19 and long COVID.

Zoom & WhatsApp Digital Information and Communication Technologies (ICTs) Enhance Community Engaged Research with Women Immigrants from Mexico.

This article demonstrates how digital information and communication technologies (ICTs) (Zoom/WhatsApp) unexpectedly and counterintuitively proved to be valuable tools for community-engaged health research when, in the context of the COVID-19 pandemic, they were integrated into a research study testing a peer support group intervention with female immigrants from Mexico. Because of pandemic restrictions, we changed the study protocol to hold meetings remotely via Zoom rather than in person as originally planned. Because we recognized that this would lack some opportunities for participants to interact and develop relationships, we created a WhatsApp chat for each group. Despite challenges for participants to use ICTs and participant-stated preference for in-person meetings, the results demonstrated that participants overwhelmingly endorsed these technologies as promoting access, participation, engagement, and satisfaction. Zoom/WhatsApp created a valuable environment both as a method for conducting research with this population, but also as part of the intervention for immigrant women to support and learn from each other. ICT adaptations have now permanently changed the way we conduct community-engaged health research.

A disrupted compartment boundary underlies abnormal cardiac patterning and congenital heart defects.

Failure of septation of the interventricular septum (IVS) is the most common congenital heart defect (CHD), but mechanisms for patterning the IVS are largely unknown. We show that a Tbx5+/Mef2cAHF+ progenitor lineage forms a compartment boundary bisecting the IVS. This coordinated population originates at a first- and second heart field interface, subsequently forming a morphogenetic nexus. Ablation of Tbx5+/Mef2cAHF+ progenitors cause IVS disorganization, right ventricular hypoplasia and mixing of IVS lineages. Reduced dosage of the CHD transcription factor TBX5 disrupts boundary position and integrity, resulting in ventricular septation defects (VSDs) and patterning defects, including Slit2 and Ntn1 misexpression. Reducing NTN1 dosage partly rescues cardiac defects in Tbx5 mutant embryos. Loss of Slit2 or Ntn1 causes VSDs and perturbed septal lineage distributions. Thus, we identify essential cues that direct progenitors to pattern a compartment boundary for proper cardiac septation, revealing new mechanisms for cardiac birth defects.

Long COVID manifests with T cell dysregulation, inflammation and an uncoordinated adaptive immune response to SARS-CoV-2.

Long COVID (LC) occurs after at least 10% of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, yet its etiology remains poorly understood. We used 'omic" assays and serology to deeply characterize the global and SARS-CoV-2-specific immunity in the blood of individuals with clear LC and non-LC clinical trajectories, 8 months postinfection. We found that LC individuals exhibited systemic inflammation and immune dysregulation. This was evidenced by global differences in T cell subset distribution implying ongoing immune responses, as well as by sex-specific perturbations in cytolytic subsets. LC individuals displayed increased frequencies of CD4+ T cells poised to migrate to inflamed tissues and exhausted SARS-CoV-2-specific CD8+ T cells, higher levels of SARS-CoV-2 antibodies and a mis-coordination between their SARS-CoV-2-specific T and B cell responses. Our analysis suggested an improper crosstalk between the cellular and humoral adaptive immunity in LC, which can lead to immune dysregulation, inflammation and clinical symptoms associated with this debilitating condition.

A foundational atlas of autism protein interactions reveals molecular convergence.

Translating high-confidence (hc) autism spectrum disorder (ASD) genes into viable treatment targets remains elusive. We constructed a foundational protein-protein interaction (PPI) network in HEK293T cells involving 100 hcASD risk genes, revealing over 1,800 PPIs (87% novel). Interactors, expressed in the human brain and enriched for ASD but not schizophrenia genetic risk, converged on protein complexes involved in neurogenesis, tubulin biology, transcriptional regulation, and chromatin modification. A PPI map of 54 patient-derived missense variants identified differential physical interactions, and we leveraged AlphaFold-Multimer predictions to prioritize direct PPIs and specific variants for interrogation in Xenopus tropicalis and human forebrain organoids. A mutation in the transcription factor FOXP1 led to reconfiguration of DNA binding sites and altered development of deep cortical layer neurons in forebrain organoids. This work offers new insights into molecular mechanisms underlying ASD and describes a powerful platform to develop and test therapeutic strategies for many genetically-defined conditions.

The APOE-R136S mutation protects against APOE4-driven Tau pathology, neurodegeneration and neuroinflammation.

Apolipoprotein E4 (APOE4) is the strongest genetic risk factor for late-onset Alzheimer's disease (LOAD), leading to earlier age of clinical onset and exacerbating pathologies. There is a critical need to identify protective targets. Recently, a rare APOE variant, APOE3-R136S (Christchurch), was found to protect against early-onset AD in a PSEN1-E280A carrier. In this study, we sought to determine if the R136S mutation also protects against APOE4-driven effects in LOAD. We generated tauopathy mouse and human iPSC-derived neuron models carrying human APOE4 with the homozygous or heterozygous R136S mutation. We found that the homozygous R136S mutation rescued APOE4-driven Tau pathology, neurodegeneration and neuroinflammation. The heterozygous R136S mutation partially protected against APOE4-driven neurodegeneration and neuroinflammation but not Tau pathology. Single-nucleus RNA sequencing revealed that the APOE4-R136S mutation increased disease-protective and diminished disease-associated cell populations in a gene dose-dependent manner. Thus, the APOE-R136S mutation protects against APOE4-driven AD pathologies, providing a target for therapeutic development against AD.

APOE4-promoted gliosis and degeneration in tauopathy are ameliorated by pharmacological inhibition of HMGB1 release.

Apolipoprotein E4 (APOE4) is an important driver of Tau pathology, gliosis, and degeneration in Alzheimer's disease (AD). Still, the mechanisms underlying these APOE4-driven pathological effects remain elusive. Here, we report in a tauopathy mouse model that APOE4 promoted the nucleocytoplasmic translocation and release of high-mobility group box 1 (HMGB1) from hippocampal neurons, which correlated with the severity of hippocampal microgliosis and degeneration. Injection of HMGB1 into the hippocampus of young APOE4-tauopathy mice induced considerable and persistent gliosis. Selective removal of neuronal APOE4 reduced HMGB1 translocation and release. Treatment of APOE4-tauopathy mice with HMGB1 inhibitors effectively blocked the intraneuronal translocation and release of HMGB1 and ameliorated the development of APOE4-driven gliosis, Tau pathology, neurodegeneration, and myelin deficits. Single-nucleus RNA sequencing revealed that treatment with HMGB1 inhibitors diminished disease-associated and enriched disease-protective subpopulations of neurons, microglia, and astrocytes in APOE4-tauopathy mice. Thus, HMGB1 inhibitors represent a promising approach for treating APOE4-related AD.

Post-acute immunological and behavioral sequelae in mice after Omicron infection.

Progress in understanding long COVID and developing effective therapeutics is hampered in part by the lack of suitable animal models. Here we used ACE2-transgenic mice recovered from Omicron (BA.1) infection to test for pulmonary and behavioral post-acute sequelae. Through in-depth phenotyping by CyTOF, we demonstrate that naïve mice experiencing a first Omicron infection exhibit profound immune perturbations in the lung after resolving acute infection. This is not observed if mice were first vaccinated with spike-encoding mRNA. The protective effects of vaccination against post-acute sequelae were associated with a highly polyfunctional SARS-CoV-2-specific T cell response that was recalled upon BA.1 breakthrough infection but not seen with BA.1 infection alone. Without vaccination, the chemokine receptor CXCR4 was uniquely upregulated on multiple pulmonary immune subsets in the BA.1 convalescent mice, a process previously connected to severe COVID-19. Taking advantage of recent developments in machine learning and computer vision, we demonstrate that BA.1 convalescent mice exhibited spontaneous behavioral changes, emotional alterations, and cognitive-related deficits in context habituation. Collectively, our data identify immunological and behavioral post-acute sequelae after Omicron infection and uncover a protective effect of vaccination against post-acute pulmonary immune perturbations.

Neuronal APOE4 removal protects against tau-mediated gliosis, neurodegeneration and myelin deficits.

Apolipoprotein E4 (APOE4) is the strongest known genetic risk factor for late-onset Alzheimer's disease (AD). Conditions of stress or injury induce APOE expression within neurons, but the role of neuronal APOE4 in AD pathogenesis is still unclear. Here we report the characterization of neuronal APOE4 effects on AD-related pathologies in an APOE4-expressing tauopathy mouse model. The selective genetic removal of APOE4 from neurons led to a significant reduction in tau pathology, gliosis, neurodegeneration, neuronal hyperexcitability and myelin deficits. Single-nucleus RNA-sequencing revealed that the removal of neuronal APOE4 greatly diminished neurodegenerative disease-associated subpopulations of neurons, oligodendrocytes, astrocytes and microglia whose accumulation correlated to the severity of tau pathology, neurodegeneration and myelin deficits. Thus, neuronal APOE4 plays a central role in promoting the development of major AD pathologies and its removal can mitigate the progressive cellular and tissue alterations occurring in this model of APOE4-driven tauopathy.

A Mesp1-dependent developmental breakpoint in transcriptional and epigenomic specification of early cardiac precursors.

Transcriptional networks governing cardiac precursor cell (CPC) specification are incompletely understood owing, in part, to limitations in distinguishing CPCs from non-cardiac mesoderm in early gastrulation. We leveraged detection of early cardiac lineage transgenes within a granular single-cell transcriptomic time course of mouse embryos to identify emerging CPCs and describe their transcriptional profiles. Mesp1, a transiently expressed mesodermal transcription factor, is canonically described as an early regulator of cardiac specification. However, we observed perdurance of CPC transgene-expressing cells in Mesp1 mutants, albeit mislocalized, prompting us to investigate the scope of the role of Mesp1 in CPC emergence and differentiation. Mesp1 mutant CPCs failed to robustly activate markers of cardiomyocyte maturity and crucial cardiac transcription factors, yet they exhibited transcriptional profiles resembling cardiac mesoderm progressing towards cardiomyocyte fates. Single-cell chromatin accessibility analysis defined a Mesp1-dependent developmental breakpoint in cardiac lineage progression at a shift from mesendoderm transcriptional networks to those necessary for cardiac patterning and morphogenesis. These results reveal Mesp1-independent aspects of early CPC specification and underscore a Mesp1-dependent regulatory landscape required for progression through cardiogenesis.

Long COVID manifests with T cell dysregulation, inflammation, and an uncoordinated adaptive immune response to SARS-CoV-2.

Long COVID (LC), a type of post-acute sequelae of SARS-CoV-2 infection (PASC), occurs after at least 10% of SARS-CoV-2 infections, yet its etiology remains poorly understood. Here, we used multiple "omics" assays (CyTOF, RNAseq/scRNAseq, Olink) and serology to deeply characterize both global and SARS-CoV-2-specific immunity from blood of individuals with clear LC and non-LC clinical trajectories, 8 months following infection and prior to receipt of any SARS-CoV-2 vaccine. Our analysis focused on deep phenotyping of T cells, which play important roles in immunity against SARS-CoV-2 yet may also contribute to COVID-19 pathogenesis. Our findings demonstrate that individuals with LC exhibit systemic inflammation and immune dysregulation. This is evidenced by global differences in T cell subset distribution in ways that imply ongoing immune responses, as well as by sex-specific perturbations in cytolytic subsets. Individuals with LC harbored increased frequencies of CD4+ T cells poised to migrate to inflamed tissues, and exhausted SARS-CoV-2-specific CD8+ T cells. They also harbored significantly higher levels of SARS-CoV-2 antibodies, and in contrast to non-LC individuals, exhibited a mis-coordination between their SARS-CoV-2-specific T and B cell responses. RNAseq/scRNAseq and Olink analyses similarly revealed immune dysregulatory mechanisms, along with non-immune associated perturbations, in individuals with LC. Collectively, our data suggest that proper crosstalk between the humoral and cellular arms of adaptive immunity has broken down in LC, and that this, perhaps in the context of persistent virus, leads to the immune dysregulation, inflammation, and clinical symptoms associated with this debilitating condition.

Single Cell Multimodal Analyses Reveal Epigenomic and Transcriptomic Basis for Birth Defects in Maternal Diabetes.

Maternal diabetes mellitus is among the most frequent environmental contributors to congenital birth defects, including heart defects and craniofacial anomalies, yet the cell types affected and mechanisms of disruption are largely unknown. Using multi-modal single cell analyses, here we show that maternal diabetes affects the epigenomic landscape of specific subsets of cardiac and craniofacial progenitors during embryogenesis. A previously unrecognized cardiac progenitor subpopulation expressing the homeodomain-containing protein ALX3 showed prominent chromatin accessibility changes and acquired a more posterior identity. Similarly, a subpopulation of neural crest-derived cells in the second pharyngeal arch, which contributes to craniofacial structures, displayed abnormalities in the epigenetic landscape and axial patterning defects. Chromatin accessibility changes in both populations were associated with increased retinoic acid signaling, known to establish anterior-posterior identity. This work highlights how an environmental insult can have highly selective epigenomic consequences on discrete cell types leading to developmental patterning defects.

Transcription Factor GATA4 Regulates Cell Type-Specific Splicing Through Direct Interaction With RNA in Human Induced Pluripotent Stem Cell-Derived Cardiac Progenitors.

BACKGROUND: GATA4 (GATA-binding protein 4), a zinc finger-containing, DNA-binding transcription factor, is essential for normal cardiac development and homeostasis in mice and humans, and mutations in this gene have been reported in human heart defects. Defects in alternative splicing are associated with many heart diseases, yet relatively little is known about how cell type- or cell state-specific alternative splicing is achieved in the heart. Here, we show that GATA4 regulates cell type-specific splicing through direct interaction with RNA and the spliceosome in human induced pluripotent stem cell-derived cardiac progenitors. METHODS: We leveraged a combination of unbiased approaches including affinity purification of GATA4 and mass spectrometry, enhanced cross-linking with immunoprecipitation, electrophoretic mobility shift assays, in vitro splicing assays, and unbiased transcriptomic analysis to uncover GATA4's novel function as a splicing regulator in human induced pluripotent stem cell-derived cardiac progenitors. RESULTS: We found that GATA4 interacts with many members of the spliceosome complex in human induced pluripotent stem cell-derived cardiac progenitors. Enhanced cross-linking with immunoprecipitation demonstrated that GATA4 also directly binds to a large number of mRNAs through defined RNA motifs in a sequence-specific manner. In vitro splicing assays indicated that GATA4 regulates alternative splicing through direct RNA binding, resulting in functionally distinct protein products. Correspondingly, knockdown of GATA4 in human induced pluripotent stem cell-derived cardiac progenitors resulted in differential alternative splicing of genes involved in cytoskeleton organization and calcium ion import, with functional consequences associated with the protein isoforms. CONCLUSIONS: This study shows that in addition to its well described transcriptional function, GATA4 interacts with members of the spliceosome complex and regulates cell type-specific alternative splicing via sequence-specific interactions with RNA. Several genes that have splicing regulated by GATA4 have functional consequences and many are associated with dilated cardiomyopathy, suggesting a novel role for GATA4 in achieving the necessary cardiac proteome in normal and stress-responsive conditions.

Association of Tinnitus with Anxiety and Depression among Active-Duty Army Personnel and Veterans at Hospital Angkatan Tentera Tuanku Mizan, Malaysia.

OBJECTIVES: To study the prevalence of tinnitus, its characteristic and its association with anxiety and depression among active-duty army personnel and veterans. This study also wants to determine the correlation of Tinnitus Handicap Inventory (THI) with Hospital Anxiety Depression Scale (HADS) among active-duty army personnel and veterans at Hospital Angkatan Tentera Tuanku Mizan. MATERIALS AND METHODS: A cross-sectional study was conducted at the Otorhinolaryngology Head and Neck Surgery Department of Hospital Angkatan Tentera Tuanku Mizan from January 2020 until June 2021, involving active-duty army personnel and veterans with at least 3 years of service. Those patients who fulfilled inclusion and exclusion criteria were recruited. Their feedback was recorded based on Malay version of Tinnitus Handicap Inventory (THI) and Malay version of Hospital Anxiety Depression Scale (HADS). RESULTS: 106 subjects responded to our questionnaires, (51 active-duty army personnel and 55 veterans) in which 4.7% (n=5) reported to have anxiety and none had depression. Overall mean score for Total THI was 27.66, suggesting that majority of our subjects only felt mild handicap due to their tinnitus. Individual THI subdomain mean scores shows that the functional subdomain (17.79) affects subjects the most as compared to the emotional (5.7) and catastrophic scores (4.21). CONCLUSION: Tinnitus can occur in all degrees of hearing loss, and it is associated with poor functional THI scores. Tinnitus is associated with anxiety but not depression among army personnel. These findings suggest that tinnitus should be addressed by healthcare providers in the military in order to maximise function and Quality of Life (QOL) among the nation's military personnel.

Differential Etv2 threshold requirement for endothelial and erythropoietic development.

Endothelial and erythropoietic lineages arise from a common developmental progenitor. Etv2 is a master transcriptional regulator required for the development of both lineages. However, the mechanisms through which Etv2 initiates the gene-regulatory networks (GRNs) for endothelial and erythropoietic specification and how the two GRNs diverge downstream of Etv2 remain incompletely understood. Here, by analyzing a hypomorphic Etv2 mutant, we demonstrate different threshold requirements for initiation of the downstream GRNs for endothelial and erythropoietic development. We show that Etv2 functions directly in a coherent feedforward transcriptional network for vascular endothelial development, and a low level of Etv2 expression is sufficient to induce and sustain the endothelial GRN. In contrast, Etv2 induces the erythropoietic GRN indirectly via activation of Tal1, which requires a significantly higher threshold of Etv2 to initiate and sustain erythropoietic development. These results provide important mechanistic insight into the divergence of the endothelial and erythropoietic lineages.

Deep immunophenotyping reveals endometriosis is marked by dysregulation of the mononuclear phagocytic system in endometrium and peripheral blood.

BACKGROUND: Endometriosis is a chronic, estrogen-dependent disorder where inflammation contributes to disease-associated symptoms of pelvic pain and infertility. Immune dysfunction includes insufficient immune lesion clearance, a pro-inflammatory endometrial environment, and systemic inflammation. Comprehensive understanding of endometriosis immune pathophysiology in different hormonal milieu and disease severity has been hampered by limited direct characterization of immune populations in endometrium, blood, and lesions. Simultaneous deep phenotyping at single-cell resolution of complex tissues has transformed our understanding of the immune system and its role in many diseases. Herein, we report mass cytometry and high dimensional analyses to study immune cell phenotypes, abundance, activation states, and functions in endometrium and blood of women with and without endometriosis in different cycle phases and disease stages. METHODS: A case-control study was designed. Endometrial biopsies and blood (n = 60 total) were obtained from women with (n = 20, n = 17, respectively) and without (n = 14, n = 9) endometriosis in the proliferative and secretory cycle phases of the menstrual cycle. Two mass cytometry panels were designed: one broad panel and one specific for mononuclear phagocytic cells (MPC), and all samples were multiplexed to characterize both endometrium and blood immune composition at unprecedented resolution. We combined supervised and unsupervised analyses to finely define the immune cell subsets with an emphasis on MPC. Then, association between cell types, protein expression, disease status, and cycle phase were performed. RESULTS: The broad panel highlighted a significant modification of MPC in endometriosis; thus, they were studied in detail with an MPC-focused panel. Endometrial CD91+ macrophages overexpressed SIRPα (phagocytosis inhibitor) and CD64 (associated with inflammation) in endometriosis, and they were more abundant in mild versus severe disease. In blood, classical and intermediate monocytes were less abundant in endometriosis, whereas plasmacytoid dendritic cells and non-classical monocytes were more abundant. Non-classical monocytes were higher in severe versus mild disease. CONCLUSIONS: A greater inflammatory phenotype and decreased phagocytic capacity of endometrial macrophages in endometriosis are consistent with defective clearance of endometrial cells shed during menses and in tissue homeostasis, with implications in endometriosis pathogenesis and pathophysiology. Different proportions of monocytes and plasmacytoid dendritic cells in blood from endometriosis suggest systemically aberrant functionality of the myeloid system opening new venues for the study of biomarkers and therapies for endometriosis.

Primary fungal laryngitis mimicking recurrent laryngeal carcinoma.

A 79-year-old smoker with a background history of a treated glottic carcinoma and chronic obstructive pulmonary disease presented with progressive hoarseness, symptoms of aspiration and shortness of breath for 6 months. Examination revealed an ulcero-fungating mass over the posterior commissure of the larynx. A tracheostomy, direct laryngoscopy and biopsy of the mass was performed to secure his airway and to exclude recurrent glottic carcinoma. Reassuringly, a histopathological examination of the mass revealed numerous fungal yeast bodies. He was then treated with itraconazole for 4 weeks and was followed up as and outpatient with complete resolution and no recurrence of the disease.