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The Journal of Allergy and Clinical... Sep 2019Thrombocytopenia is a serious issue for all patients with classical Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) because it causes severe and...
BACKGROUND
Thrombocytopenia is a serious issue for all patients with classical Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT) because it causes severe and life-threatening bleeding. Lentiviral gene therapy (GT) for WAS has shown promising results in terms of immune reconstitution. However, despite the reduced severity and frequency of bleeding events, platelet counts remain low in GT-treated patients.
OBJECTIVE
We carefully investigated platelet defects in terms of phenotype and function in untreated patients with WAS and assessed the effect of GT treatment on platelet dysfunction.
METHODS
We analyzed a cohort of 20 patients with WAS/XLT, 15 of them receiving GT. Platelet phenotype and function were analyzed by using electron microscopy, flow cytometry, and an aggregation assay. Platelet protein composition was assessed before and after GT by means of proteomic profile analysis.
RESULTS
We show that platelets from untreated patients with WAS have reduced size, abnormal ultrastructure, and a hyperactivated phenotype at steady state, whereas activation and aggregation responses to agonists are decreased. GT restores platelet size and function early after treatment and reduces the hyperactivated phenotype proportionally to WAS protein expression and length of follow-up.
CONCLUSIONS
Our study highlights the coexistence of morphologic and multiple functional defects in platelets lacking WAS protein and demonstrates that GT normalizes the platelet proteomic profile with consequent restoration of platelet ultrastructure and phenotype, which might explain the observed reduction of bleeding episodes after GT. These results are instrumental also from the perspective of a future clinical trial in patients with XLT only presenting with microthrombocytopenia.
Topics: Adolescent; Adult; Blood Platelets; Child; Child, Preschool; Female; Genetic Therapy; Hematopoietic Stem Cell Transplantation; Humans; Infant; Lentivirus; Male; Microscopy, Electron, Transmission; Phenotype; Platelet Activation; Platelet Count; Wiskott-Aldrich Syndrome; Wiskott-Aldrich Syndrome Protein
PubMed: 30926529
DOI: 10.1016/j.jaci.2019.03.012 -
IScience Jan 2022The Wiskott-Aldrich syndrome protein and SCAR homolog (WASH), an actin nucleation-promoting factor, is present in the nucleus where it regulates gene transcription and...
The Wiskott-Aldrich syndrome protein and SCAR homolog (WASH), an actin nucleation-promoting factor, is present in the nucleus where it regulates gene transcription and maintains nuclear organization. Here, we show that WASH interacts with core non-homologous end-joining (NHEJ) factors including Ku70/Ku80 and DNA-PKcs, and Ku70/Ku80 is involved in the recruitment of WASH to the sites of DNA double-stranded break (DSB). WASH depletion leads to increased cell sensitivity and impaired DNA repair capacity in response to etoposide-induced DSBs and reduces NHEJ efficiency. Mechanistically, we show that loss of WASH inhibits the phosphorylation of DNA-PKcs, H2AX, and KAP1 after DSB induction and reduces chromatin relaxation and the recruitment of several downstream NHEJ factors to DSBs. Moreover, WASH role in DSB repair depends on its conserved C-terminal VCA domain and Arp2/3 activation. Our findings reveal a function and mechanistic insight for WASH in DNA DSB repair by the NHEJ pathway.
PubMed: 35036867
DOI: 10.1016/j.isci.2021.103676 -
Small GTPases Nov 2019The Arf and Rho subfamilies of small GTPases are nucleotide-dependent molecular switches that act as master regulators of vesicular trafficking and the actin... (Review)
Review
The Arf and Rho subfamilies of small GTPases are nucleotide-dependent molecular switches that act as master regulators of vesicular trafficking and the actin cytoskeleton organization. Small GTPases control cell processes with high fidelity by acting through distinct repertoires of binding partners called effectors. While we understand a great deal about how these GTPases act individually, relatively little is known about how they cooperate, especially in the control of effectors. This review highlights how Arf GTPases collaborate with Rac1 to regulate actin cytoskeleton dynamics at the membrane via recruiting and activating the Wave Regulatory Complex (WRC), a Rho effector that underpins lamellipodia formation and macropinocytosis. This provides insight into Arf regulation of the actin cytoskeleton, while putting the spotlight on small GTPase cooperation with emerging evidence of its importance in fundamental cell biology and interactions with pathogenic bacteria.
Topics: ADP-Ribosylation Factors; Actin Cytoskeleton; Animals; Escherichia coli; Humans; Salmonella; Wiskott-Aldrich Syndrome Protein Family; rho GTP-Binding Proteins
PubMed: 28524754
DOI: 10.1080/21541248.2017.1329691 -
The World Allergy Organization Journal Jul 2020Primary Immunodeficiency Disorders (PIDs) are well-known disorders in the West. but the recognition and diagnosis of these disorders is challenging in developing...
BACKGROUND
Primary Immunodeficiency Disorders (PIDs) are well-known disorders in the West. but the recognition and diagnosis of these disorders is challenging in developing countries. We present the spectrum of PIDs seen at a tertiary care center in Pakistan, identified using clinical case definitions and molecular methods.
METHODS
A retrospective chart review of children suspected to have PID was conducted at the Aga Khan University Hospital (AKUH) Karachi, Pakistan from 2010 to 2016. Data on demographics, clinical features, family history of consanguinity, sibling death, details of laboratory workup done for PID and molecular tests targeted panel next generation sequencing (NGS) or whole exome sequencing (WES) performed at the Geha laboratory at Boston Children's Hospital, USA was collected. The study was exempted from the Ethical Review Committee of AKUH.
RESULTS
A total of 43 children visited the hospital with suspected PID during the study period. Genetic testing was performed in 31/43 (72.1%) children. A confirmed diagnosis of PID was established in 20/43 (46.5%) children. A pathogenic gene variant was identified in 17(85%) of the 20 confirmed cases (Table 1). Twelve (60%) of the confirmed cases of PID were male. The most common presenting symptom was recurrent diarrhea 11/20 (55%). The mean (±S.D) age of the cases at the time of diagnosis was 4.2 (±4.1) years. Chronic granulomatous disease (CGD) was the most common 6/20 (30%) disorder, followed by severe combined immunodeficiency (SCID) 3/20 (15%), leukocyte adhesion deficiency (LAD) 3/20 (15%), agammaglobulinemia/hypogammaglobulinemia 3/20 (15%), and Hermansky-Pudlak Syndrome (HPS) 2/20 (10%). Wiskott-Aldrich Syndrome, Immunodeficiency Centromeric Instability and Facial Anomalies Syndrome (ICF 2), Trichohepatoenteric syndrome (TRES), and C3 deficiency were each diagnosed once {1/20 (4.3%) each} (Table 1). Of these 20 confirmed cases, almost all 19/20 (95%) had a family history of consanguinity. Sibling death was reported in 5/20 (25%) of these cases. Five out of the 20 (25%) children died over the 7-year period for various reasons.
CONCLUSION
PIDs are not uncommon in Pakistan; their diagnosis may be missed or delayed due to the overlapping of clinical features of PID with other diseases and a lack of diagnostic facilities. There is a need to build capacity for early recognition and diagnosis of PIDs to decrease morbidity and mortality.
PubMed: 32793328
DOI: 10.1016/j.waojou.2020.100133 -
Blood Advances Dec 2022Mature bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in this process can lead to...
Mature bone marrow (BM) megakaryocytes (MKs) produce platelets by extending proplatelets into sinusoidal blood vessels. Defects in this process can lead to thrombocytopenia and increased risk of bleeding. Mice lacking the actin-regulatory proteins Profilin 1 (PFN1), Wiskott-Aldrich Syndrome protein (WASp), Actin Related Protein 2/3 complex (Arp2/3), or adhesion and degranulation-promoting adapter protein (ADAP) display thrombocytopenia and ectopic release of (pro)platelet-like particles into the BM compartment, pointing to an important axis of actin-mediated directional proplatelet formation. The mechanism underlying ectopic release in these mice is still not completely understood. However, we hypothesized that similar functional defects account for this observation. We analyzed WASp-, ADAP-, PFN1-, and ARPC2-knockout mice to determine the role of actin reorganization and integrin activation in directional proplatelet formation. ADAP-, ARPC2-, and PFN1-deficient MKs displayed reduced adhesion to collagen, defective F-actin organization, and diminished β1-integrin activation. WASp-deficient MKs showed the strongest reduction in the adhesion assay of collagen and altered F-actin organization with reduced podosome formation. Our results indicate that ADAP, PFN1, WASp, and ARP2/3 are part of the same pathway that regulates polarization processes in MKs and directional proplatelet formation into BM sinusoids.
Topics: Mice; Animals; Megakaryocytes; Actins; Thrombocytopenia; Mice, Knockout; Disease Models, Animal; Adaptor Proteins, Signal Transducing; Integrins
PubMed: 36251748
DOI: 10.1182/bloodadvances.2022007824 -
JCI Insight May 2020Several studies have suggested an oncogenic role for the neural Wiskott-Aldrich syndrome protein (N-WASP, encoded by the Wasl gene), but thus far, little is known about...
Several studies have suggested an oncogenic role for the neural Wiskott-Aldrich syndrome protein (N-WASP, encoded by the Wasl gene), but thus far, little is known about its function in pancreatic ductal adenocarcinoma (PDAC). In this study, we performed in silico analysis of WASL expression in PDAC patients and found a correlation between low WASL expression and prolonged survival. To clarify the role of Wasl in pancreatic carcinogenesis, we used 2 oncogenic Kras-based PDAC mouse models with pancreas-specific Wasl deletion. In line with human data, both mouse models had an increased survival benefit due to either impaired tumor development in the presence of the tumor suppressor Trp53 or the delayed tumor progression and senescent phenotype upon genetic ablation of Trp53. Mechanistically, loss of Wasl resulted in cell-autonomous senescence through displacement of the N-WASP binding partners WASP-interacting protein (WIP) and p120ctn; vesicular accumulation of GSK3β, as well as YAP1 and phosphorylated β-catenin, which are components of the destruction complex; and upregulation of Cdkn1a(p21), a master regulator of senescence. Our findings, thus, indicate that Wasl functions in an oncogenic manner in PDAC by promoting the deregulation of the p120-catenin/β-catenin/p21 pathway. Therefore, strategies to reduce N-WASP activity might improve the survival outcomes of PDAC patients.
Topics: Animals; Humans; Mice; Mice, Transgenic; Neoplasms, Experimental; Pancreatic Neoplasms; Tumor Suppressor Protein p53; Wiskott-Aldrich Syndrome Protein, Neuronal
PubMed: 32434991
DOI: 10.1172/jci.insight.127275 -
Revista Chilena de Pediatria Feb 2020Guillain-Barre Syndrome (GBS) is rarely diagnosed in the first year of life. The association of GBS with Wiskott-Aldrich syndrome (WAS) is even less frequent and has...
INTRODUCTION
Guillain-Barre Syndrome (GBS) is rarely diagnosed in the first year of life. The association of GBS with Wiskott-Aldrich syndrome (WAS) is even less frequent and has been previously reported in only two children to our knowledge. Hydrocephalus is a known but rare complication of GBS.
OBJECTIVE
To describe the case of an infant in which GBS, WAS and hydrocephalus appear clinically associated.
CLINICAL CASE
A nine-months-old male infant with a history of WAS was admitted to our ICU with acute hypotonia and poor general condition. He developed flaccid paralysis, absent deep tendon reflexes, and respiratory failure. A lumbar puncture showed albuminocytologic dissociation. GBS was suspected and an electromyography was performed, showing a demyelinating polyneuropathy. He was successfully treated with intravenous immunoglobulins. During hospitalization, he presented intermittent bradycardia, so a brain CT scan was performed, showing acute hydrocephalus which was managed through an external ventricular drain achieving favorable response. In the long term, the patient underwent bone marrow transplant and had to be reoperated due to valve-related complications. However, his psychomotor development is normal, with no obvious neurological sequelae.
CONCLUSION
We present the third case of GBS in a patient with WAS, which is the first infant younger than one year. Additionally, he presented acute hydrocephalus as a complication of GBS. We suggest considering these three comorbidities since their early diagnosis and prompt management allow bet ter neurological recovery and avoid potentially lethal complications.
Topics: Guillain-Barre Syndrome; Humans; Hydrocephalus; Infant; Male; Wiskott-Aldrich Syndrome
PubMed: 32730420
DOI: 10.32641/rchped.v91i1.1208 -
Mucosal Immunology Feb 2024Dysregulated B cell responses have been described in inflammatory bowel disease (IBD) patients; however, the role of B cells in IBD pathology remained incompletely...
Dysregulated B cell responses have been described in inflammatory bowel disease (IBD) patients; however, the role of B cells in IBD pathology remained incompletely understood. We here provide evidence for the detrimental role of activated B cells during the onset of autoimmune intestinal inflammation. Using Wiskott-Aldrich Syndrome interacting protein deficient (Wipf1) mice as a mouse model of chronic colitis, we identified clusters of differentiation (CD)86 expression on activated B cells as a crucial factor exacerbating pro-inflammatory cytokine production of intestinal CD4 T cells. Depleting B cells through anti-CD20 antibody treatment or blocking costimulatory signals mediated by CD86 through cytotoxic T lymphocyte antigen-4-immunoglobulin (CTLA-4-Ig) diminished intestinal inflammation in our mouse model of chronic IBD at the onset of disease. This was due to a reduction in aberrant humoral immune responses and reduced CD4 T cell pro-inflammatory cytokine production, especially interferon-g (IFN-g) and granulocyte-macrophage colony-stimulating factor (GM-CSF). Interestingly, in addition to B cells isolated from the inflamed colon of Wipf1 mice, we also found CD86 mRNA and protein expression upregulated on activated B cells isolated from inflamed tissue of human patients with IBD. B cell activation and CD86 expression were boosted by soluble CD40L in vitro, which we found in the serum of mice and human patients with IBD. In summary, our data provides detailed insight into the contribution of B cells to intestinal inflammation, with implications for the treatment of IBD.
Topics: Humans; CD4-Positive T-Lymphocytes; Colitis; Inflammation; Inflammatory Bowel Diseases; Intestinal Mucosa; Intestines
PubMed: 37918715
DOI: 10.1016/j.mucimm.2023.10.005 -
Scientific Reports Mar 2023Rho family GTPases regulate both linear and branched actin dynamics by activating downstream effectors to facilitate the assembly and function of complex cellular...
Rho family GTPases regulate both linear and branched actin dynamics by activating downstream effectors to facilitate the assembly and function of complex cellular structures such as lamellipodia and contractile actomyosin rings. Wiskott-Aldrich Syndrome (WAS) family proteins are downstream effectors of Rho family GTPases that usually function in a one-to-one correspondence to regulate branched actin nucleation. In particular, the WAS protein Scar/WAVE has been shown to exhibit one-to-one correspondence with Rac GTPase. Here we show that Rac and SCAR are recruited to cell wounds in the Drosophila repair model and are required for the proper formation and maintenance of the dynamic actomyosin ring formed at the wound periphery. Interestingly, we find that SCAR is recruited to wounds earlier than Rac and is still recruited to the wound periphery in the presence of a potent Rac inhibitor. We also show that while Rac is important for actin recruitment to the actomyosin ring, SCAR serves to organize the actomyosin ring and facilitate its anchoring to the overlying plasma membrane. These differing spatiotemporal recruitment patterns and wound repair phenotypes highlight the Rac-independent functions of SCAR and provide an exciting new context in which to investigate these newly uncovered SCAR functions.
Topics: Animals; Actins; Actomyosin; Cytokinesis; Actin Cytoskeleton; Drosophila; rho GTP-Binding Proteins; Cicatrix; Wiskott-Aldrich Syndrome Protein Family; rac GTP-Binding Proteins
PubMed: 36959278
DOI: 10.1038/s41598-023-31973-2 -
Frontiers in Immunology 2021The T cell receptor (TCR) diversity is essential for effective T cell immunity. Previous studies showed that TCR diversity in Wiskott-Aldrich Syndrome (WAS) patients was...
BACKGROUND
The T cell receptor (TCR) diversity is essential for effective T cell immunity. Previous studies showed that TCR diversity in Wiskott-Aldrich Syndrome (WAS) patients was severely impaired, especially in the memory T cell populations. Whether this defect was caused by intrinsic WASp deficiency or extrinsic reasons is still unclear.
METHODS
We sorted different T cell subsets from the bone marrow chimeric mice model using both magnetic beads and flow cytometry. TCR repertoires of memory T cells, especially CD4 effector memory T (TEM) cells and CD8 central memory T (TCM) cells, were analyzed using the UMI quantitative high-throughput sequencing (HTS).
RESULTS
An average of 5.51 million sequencing reads of 32 samples was obtained from the Illumina sequencing platform. Bioinformatic analyses showed that compared with wild type (WT), WAS knock out (KO)-CD4 TEM cells exhibited increased Simpson index and decreased D50 index (P <0.05); The rank abundance curve of KO-CD4 TEM cells was shorter and steeper than that of WT, and the angle of D and q in KO-CD4 TEM cells was lower than that of WT, while these indexes showed few changes between WT and KO chimeric mice in the CD8TCM population. Therefore, it indicated that the restriction on the TCRVβ repertoires is majorly in KO-CD4 TEM cells but not KO- CD8 TCM cells. Principal Component Analysis (PCA), a comprehensive parameter for TCRVβ diversity, successfully segregated CD4 TEM cells from WT and KO, but failed in CD8 TCM cells. Among the total sequences of , the usage of TRBV12.2, TRBV30, TRBV31, TRBV4, TRBD1, TRBD2, TRBJ1.1, and TRBJ1.4 showed a significant difference between WT-CD4 TEM cells and KO-CD4 TEM cells (P <0.05), while in CD8 TCM cells, only the usage of TRBV12.2 and TRBV20 showed a substantial difference between WT and KO (P <0.05). No significant differences in the hydrophobicity and sequence length of TCRVβ were found between the WT and KO groups.
CONCLUSION
WASp deficiency selectively affected the TCR diversity of different memory T cell subsets, and it had more impact on the TCRVβ diversity of CD4 TEM cells than CD8 TCM cells. Moreover, the limitation of TCRVβ diversity of CD4 TEM cells and CD8 TCM cells in WAS was not severe but intrinsic.
Topics: Amino Acid Sequence; Animals; Bone Marrow Transplantation; Computational Biology; Disease Models, Animal; Genetic Variation; High-Throughput Nucleotide Sequencing; Hydrophobic and Hydrophilic Interactions; Memory T Cells; Mice; Receptors, Antigen, T-Cell; T-Lymphocyte Subsets; Transplantation Chimera; V(D)J Recombination; Wiskott-Aldrich Syndrome
PubMed: 35116029
DOI: 10.3389/fimmu.2021.794795