Manish Butte

Epithelial requirement for in vitro proliferation and xenograft growth and metastasis of MDA-MB-468 human breast cancer cells: oncogenic rather than tumor-suppressive role of E-cadherin.

Breast Cancer Res. 2017 Jul 27;19(1):86

Authors: Hugo HJ, Gunasinghe NPAD, Hollier BG, Tanaka T, Blick T, Toh A, Hill P, Gilles C, Waltham M, Thompson EW

Abstract
BACKGROUND: Epithelial-to-mesenchymal transition (EMT) is associated with downregulated E-cadherin and frequently with decreased proliferation. Proliferation may be restored in secondary metastases by mesenchymal-to-epithelial transition (MET). We tested whether E-cadherin maintains epithelial proliferation in MDA-MB-468 breast cancer cells, facilitating metastatic colonization in severe combined immunodeficiency (SCID) mice.
METHODS: EMT/MET markers were assessed in xenograft tumors by immunohistochemistry. Stable E-cadherin manipulation was effected by transfection and verified by Western blotting, immunocytochemistry, and quantitative polymerase chain reaction (qPCR). Effects of E-cadherin manipulation on proliferation and chemomigration were assessed in vitro by performing sulforhodamine B assays and Transwell assays, respectively. Invasion was assessed by Matrigel outgrowth; growth in vivo was assessed in SCID mice; and EMT status was assessed by qPCR. Hypoxic response of E-cadherin knockdown cell lines was assessed by qPCR after hypoxic culture. Repeated measures analysis of variance (ANOVA), one- and two-way ANOVA with posttests, and paired Student’s t tests were performed to determine significance (p < 0.05).
RESULTS: EMT occurred at the necrotic interface of MDA-MB-468 xenografts in regions of hypoxia. Extratumoral deposits (vascular and lymphatic inclusions, local and axillary nodes, and lung metastases) strongly expressed E-cadherin. MDA-MB-468 cells overexpressing E-cadherin were more proliferative and less migratory in vitro, whereas E-cadherin knockdown (short hairpin CDH1 [shCDH1]) cells were more migratory and invasive, less proliferative, and took longer to form tumors. shCDH1-MDA-MB-468 xenografts did not contain the hypoxia-induced necrotic areas observed in wild-type (WT) and shSCR-MDA-MB-468 tumors, but they did not exhibit an impaired hypoxic response in vitro. Although vimentin expression was not stimulated by E-cadherin knockdown in 2D or 3D cultures, xenografts of these cells were globally vimentin-positive rather than exhibiting regional EMT, and they expressed higher SNA1 than their in vitro counterparts. E-cadherin suppression caused a trend toward reduced lung metastasis, whereas E-cadherin overexpression resulted in the reverse trend, consistent with the increased proliferation rate and predominantly epithelial phenotype of MDA-MB-468 cells outside the primary xenograft. This was also originally observed in WT xenografts. Furthermore, we found that patients with breast cancer that expressed E-cadherin were more likely to have metastases.
CONCLUSIONS: E-cadherin expression promotes growth of primary breast tumors and conceivably the formation of metastases, supporting a role for MET in metastasis. E-cadherin needs to be reevaluated as a tumor suppressor.

PMID: 28750639 [PubMed – in process]

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Family History of Early Infant Death Correlates with Earlier Age at Diagnosis But Not Shorter Time to Diagnosis for Severe Combined Immunodeficiency.

Front Immunol. 2017;8:808

Authors: Luk ADW, Lee PP, Mao H, Chan KW, Chen XY, Chen TX, He JX, Kechout N, Suri D, Tao YB, Xu YB, Jiang LP, Liew WK, Jirapongsananuruk O, Daengsuwan T, Gupta A, Singh S, Rawat A, Abdul Latiff AH, Lee ACW, Shek LP, Nguyen TVA, Chin TJ, Chien YH, Latiff ZA, Le TMH, Le NNQ, Lee BW, Li Q, Raj D, Barbouche MR, Thong MK, Ang MCD, Wang XC, Xu CG, Yu HG, Yu HH, Lee TL, Yau FYS, Wong WH, Tu W, Yang W, Chong PCY, Ho MHK, Lau YL

Abstract
BACKGROUND: Severe combined immunodeficiency (SCID) is fatal unless treated with hematopoietic stem cell transplant. Delay in diagnosis is common without newborn screening. Family history of infant death due to infection or known SCID (FH) has been associated with earlier diagnosis.
OBJECTIVE: The aim of this study was to identify the clinical features that affect age at diagnosis (AD) and time to the diagnosis of SCID.
METHODS: From 2005 to 2016, 147 SCID patients were referred to the Asian Primary Immunodeficiency Network. Patients with genetic diagnosis, age at presentation (AP), and AD were selected for study.
RESULTS: A total of 88 different SCID gene mutations were identified in 94 patients, including 49 IL2RG mutations, 12 RAG1 mutations, 8 RAG2 mutations, 7 JAK3 mutations, 4 DCLRE1C mutations, 4 IL7R mutations, 2 RFXANK mutations, and 2 ADA mutations. A total of 29 mutations were previously unreported. Eighty-three of the 94 patients fulfilled the selection criteria. Their median AD was 4 months, and the time to diagnosis was 2 months. The commonest SCID was X-linked (n = 57). A total of 29 patients had a positive FH. Candidiasis (n = 27) and bacillus Calmette-Guérin (BCG) vaccine infection (n = 19) were the commonest infections. The median age for candidiasis and BCG infection documented were 3 months and 4 months, respectively. The median absolute lymphocyte count (ALC) was 1.05 × 10(9)/L with over 88% patients below 3 × 10(9)/L. Positive FH was associated with earlier AP by 1 month (p = 0.002) and diagnosis by 2 months (p = 0.008), but not shorter time to diagnosis (p = 0.494). Candidiasis was associated with later AD by 2 months (p = 0.008) and longer time to diagnosis by 0.55 months (p = 0.003). BCG infections were not associated with age or time to diagnosis.
CONCLUSION: FH was useful to aid earlier diagnosis but was overlooked by clinicians and not by parents. Similarly, typical clinical features of SCID were not recognized by clinicians to shorten the time to diagnosis. We suggest that lymphocyte subset should be performed for any infant with one or more of the following four clinical features: FH, candidiasis, BCG infections, and ALC below 3 × 10(9)/L.

PMID: 28747913 [PubMed]

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Acquired Senescent T-Cell Phenotype Correlates with Clinical Severity in GATA Binding Protein 2-Deficient Patients.

Front Immunol. 2017;8:802

Authors: Ruiz-García R, Rodríguez-Vigil C, Marco FM, Gallego-Bustos F, Castro-Panete MJ, Diez-Alonso L, Muñoz-Ruiz C, Ruiz-Contreras J, Paz-Artal E, González-Granado LI, Allende LM

Abstract
GATA binding protein 2 (GATA2) deficiency is a rare disorder of hematopoiesis, lymphatics, and immunity caused by spontaneous or autosomal dominant mutations in the GATA2 gene. Clinical manifestations range from neutropenia, lymphedema, deafness, to severe viral and mycobacterial infections, bone marrow failure, and acute myeloid leukemia. Patients also present with monocytopenia, dendritic cell, B- and natural killer (NK)-cell deficiency. We studied the T-cell and NK-cell compartments of four GATA2-deficient patients to assess if changes in these lymphocyte populations could be correlated with clinical phenotype. Patients with more severe clinical complications demonstrated a senescent T-cell phenotype whereas patients with lower clinical score had undetectable changes relative to controls. In contrast, patients’ NK-cells demonstrated an immature/activated phenotype that did not correlate with clinical score, suggesting an intrinsic NK-cell defect. These studies will help us to determine the contribution of T- and NK-cell dysregulation to the clinical phenotype of GATA2 patients, and may help to establish the most accurate therapeutic options for these patients. Asymptomatic patients may be taken into consideration for hematopoietic stem cell transplantation when dysregulation of T-cell and NK-cell compartment is present.

PMID: 28747912 [PubMed]

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IFN-γR1 defects: Mutation update and description of the IFNGR1 variation database.

Hum Mutat. 2017 Jul 25;:

Authors: van de Vosse E, van Dissel JT

Abstract
IFN-γ signaling is essential for the innate immune defense against mycobacterial infections. IFN-γ signals through the IFN-γ receptor which consists of a tetramer of two IFN-γR1 chains in complex with two IFN-γR2 chains, where IFN-γR1 is the ligand-binding chain of the interferon-γ receptor and IFN-γR2 is the signal-transducing chain of the IFN-γ receptor. Germline mutations in the gene IFNGR1 encoding the IFN-γR1 cause a primary immunodeficiency that mainly leads to mycobacterial infections. Here, we review the molecular basis of this immunodeficiency in the 130 individuals described to date, and report mutations in 5 new individuals, bringing the total number to 135 individuals from 98 kindreds. 40 unique IFNGR1 mutations have been reported and they exert either an autosomal dominant or an autosomal recessive effect. Mutations resulting in premature stopcodons represent the majority of IFNGR1 mutations (60%; 24/40), followed by amino acid substitutions (28%, 11/40). All known mutations, as well as 287 other variations, have been deposited in the online IFNGR1 variation database (www.LOVD.nl/IFNGR1). In this article, we review the function of IFN-γR1 and molecular genetics of human IFNGR1. This article is protected by copyright. All rights reserved.

PMID: 28744922 [PubMed – as supplied by publisher]

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Analysis of the recovery of CD247 expression in a PID patient: Insights into the spontaneous repair of defective genes.

Blood. 2017 Jul 25;:

Authors: Blázquez-Moreno A, Pérez-Portilla A, Agúndez-Llaca M, Dukovska D, Valés-Gómez M, Aydogmus C, Ikinciogullari A, Regueiro JR, Reyburn HT

Abstract
Mutations in T-cell antigen receptor (TCR) subunits genes cause rare immunodeficiency diseases characterized by impaired expression of the TCR at the cell surface and selective T lymphopenia. Here detailed analyses of spontaneously arising somatic mutations that recover CD247, and so TCR expression, in a newly identified CD247 deficient patient are described. The recovery of CD247 expression in some patient T cells was associated with both reversion of the inactivating mutation and a variant with a compensating mutation that could reconstitute TCR expression, but not as efficiently as wild-type CD247. Multiple mutations were found in CD247 cDNAs cloned from the patient as well as in cDNA and genomic DNA from other individuals suggesting that genetic variation in this gene is frequent. Analyses of other genes mutated in primary immunodeficiencies where reversions have been described also revealed a higher rate of mutation than that observed for genes mutated in immunodeficiency where revertants have not been identified or control genes. These data support the hypothesis that the occurrence of somatic mutations that may reconstitute genetic defects in primary immunodeficiency is related to an increased propensity of those genes to mutate.

PMID: 28743717 [PubMed – as supplied by publisher]

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Loss of NHEJ1 Protein Due to a Novel Splice Site Mutation in a Family Presenting with Combined Immunodeficiency, Microcephaly, and Growth Retardation and Literature Review.

J Clin Immunol. 2017 Jul 24;:

Authors: Sheikh F, Hawwari A, Alhissi S, Al Gazlan S, Al Dhekri H, Rehan Khaliq AM, Borrero E, El-Baik L, Arnaout R, Al-Mousa H, Alazami AM

Abstract
INTRODUCTION: Non-homologous end joining gene 1 (NHEJ1) defect is a rare form of primary immune deficiency. Very few cases have been described from around the world.
PURPOSE: We are reporting the first family from the Arabian Gulf with three siblings presenting with combined immunodeficiency (CID), microcephaly, and growth retardation due to a novel NHEJ1 splice site mutation, in addition to a review of the previously published literature on this subject.
METHODS: Patients’ clinical, immunological, and laboratory features were examined. Samples were subjected to targeted next-generation sequencing (NGS). The pathogenic change in NHEJ1 was confirmed by Sanger sequencing, then further assessed at the RNA and protein levels.
RESULTS: Patients were found to have a homozygous splice site mutation immediately downstream of exon 3 in NHEJ1 (c.390 + 1G > C). This led to two distinct mRNA products, one of which demonstrated skipping of the last 69 basepairs (bp) of exon 3 while the other showed complete skipping of the entire exon. Although both deletions were in-frame, immunoblotting did not reveal any NHEJ1 protein products in patient cells, indicating a null phenotype.
CONCLUSION: Patients presenting with CID, microcephaly, and growth retardation should be screened for NHEJ1 gene mutations. We discuss our data in the context of one of our patients who is still alive at the age of 30 years, without transplantation, and who is the longest known survivor of this disease.

PMID: 28741180 [PubMed – as supplied by publisher]

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Autoimmunity and its association with regulatory T cells and B cell subsets in patients with common variable immunodeficiency.

Allergol Immunopathol (Madr). 2017 Jul 20;:

Authors: Azizi G, Abolhassani H, Kiaee F, Tavakolinia N, Rafiemanesh H, Yazdani R, Mahdaviani SA, Mohammadikhajehdehi S, Tavakol M, Ziaee V, Negahdari B, Mohammadi J, Mirshafiey A, Aghamohammadi A

Abstract
BACKGROUND: Common variable immunodeficiency (CVID) is one of the most prevalent symptomatic primary immunodeficiencies (PIDs), which manifests a wide clinical variability such as autoimmunity, as well as T cell and B cell abnormalities.
METHODS: A total of 72 patients with CVID were enrolled in this study. Patients were evaluated for clinical manifestations and classified according to the presence or absence of autoimmune disease. We measured regulatory T cells (Tregs) and B-cell subsets using flow cytometry, as well as specific antibody response (SAR) to pneumococcal vaccine, autoantibodies and anti-IgA in patients.
RESULTS: Twenty-nine patients (40.3%) have shown at least one autoimmune manifestation. Autoimmune cytopenias and autoimmune gastrointestinal diseases were the most common. A significant association was detected between autoimmunity and presence of hepatomegaly and splenomegaly. Among CVID patients, 38.5% and 79.3% presented a defect in Tregs and switched memory B-cells, respectively, whereas 69.0% presented CD21(low) B cell expansion. Among patients with a defect in Treg, switched memory and CD21(low) B cell, the frequency of autoimmunity was 80.0%, 52.2% and 55.0%, respectively. A negative correlation was observed between the frequency of Tregs and CD21(low) B cell population. 82.2% of patients had a defective SAR which was associated with the lack of autoantibodies.
CONCLUSIONS: Autoimmunity may be the first clinical manifestation of CVID, thus routine screening of immunoglobulins is suggested for patients with autoimmunity. Lack of SAR in CVID is associated with the lack of specific autoantibodies in patients with autoimmunity. It is suggested that physicians use alternative diagnostic procedures.

PMID: 28735808 [PubMed – as supplied by publisher]

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Respiratory Infections and Antibiotic Usage in Common Variable Immunodeficiency.

J Allergy Clin Immunol Pract. 2017 Jul 19;:

Authors: Sperlich JM, Grimbacher B, Workman S, Haque T, Seneviratne SL, Burns SO, Reiser V, Vach W, Hurst JR, Lowe DM

Abstract
BACKGROUND: Patients with common variable immunodeficiency (CVID) suffer frequent respiratory tract infections despite immunoglobulin replacement and are prescribed significant quantities of antibiotics. The clinical and microbiological nature of these exacerbations, the symptomatic triggers to take antibiotics, and the response to treatment have not been previously investigated.
OBJECTIVES: To describe the nature, frequency, treatment, and clinical course of respiratory tract exacerbations in patients with CVID and to describe pathogens isolated during respiratory tract exacerbations.
METHODS: We performed a prospective diary card exercise in 69 patients with CVID recruited from a primary immunodeficiency clinic in the United Kingdom, generating 6210 days of symptom data. We collected microbiology (sputum microscopy and culture, atypical bacterial PCR, and mycobacterial culture) and virology (nasopharyngeal swab multiplex PCR) samples from symptomatic patients with CVID.
RESULTS: There were 170 symptomatic exacerbations and 76 exacerbations treated by antibiotics. The strongest symptomatic predictors for commencing antibiotics were cough, shortness of breath, and purulent sputum. There was a median delay of 5 days from the onset of symptoms to commencing antibiotics. Episodes characterized by purulent sputum responded more quickly to antibiotics, whereas sore throat and upper respiratory tract symptoms responded less quickly. A pathogenic virus was isolated in 56% of respiratory exacerbations and a potentially pathogenic bacteria in 33%.
CONCLUSIONS: Patients with CVID delay and avoid treatment of symptomatic respiratory exacerbations, which could result in structural lung damage. However, viruses are commonly represented and illnesses dominated by upper respiratory tract symptoms respond poorly to antibiotics, suggesting that antibiotic usage could be better targeted.

PMID: 28734862 [PubMed – as supplied by publisher]

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EASIX in patients with acute graft-versus-host disease: a retrospective cohort analysis.

Lancet Haematol. 2017 Jul 18;:

Authors: Luft T, Benner A, Jodele S, Dandoy CE, Storb R, Gooley T, Sandmaier BM, Becker N, Radujkovic A, Dreger P, Penack O

Abstract
BACKGROUND: Endothelial dysfunction links thrombotic microangiopathy to steroid-refractory graft-versus-host disease (GVHD) after allogeneic stem-cell transplantation. We aimed to assess if the simple formula-lactate dehydrogenase (U/L) × creatinine (mg/dL)/thrombocytes (10(9) cells per L)-termed the Endothelial Activation and Stress Index (EASIX), might be valuable for the prediction of death in patients with acute GVHD after allogeneic stem-cell transplantation.
METHODS: For this retrospective analysis, we analysed a training cohort (in Germany) and three validation cohorts (in Germany and the USA) of patients with acute GVHD who had received consecutive allogeneic stem-cell transplantation. The primary endpoint was prediction of overall survival when measured at acute GVHD onset (EASIX-GVHD). We validated the prognostic strength of EASIX-GVHD for overall survival and non-relapse mortality in the three independent cohorts by calculating the prediction error (integrated Brier score), and concordance index.
FINDINGS: In the total cohort of patients with acute GVHD (n=311), EASIX-GVHD predicted overall survival in univariable and multivariable models (univariate analysis, hazard ratio [HR] for a one-fold increase 1·16, 95% CI 1·12-1·20, p=0·0004). However, in the subpopulation of patients with myeloablative conditioning (n=72), EASIX-GVHD did not predict overall survival, which is probably attributable to thrombocytopenia at GVHD onset (73 × 10(9) cells per L [IQR 29·75-180·00] for myeloablative conditioning vs 160 × 10(9) cells per L [90·0-250·5] for reduced-intensity conditioning; p<0·0001). In patients who received reduced-intensity conditioning (n=239), EASIX-GVHD was a strong predictor of overall survival (HR for a two-fold change of 1·23, 95% CI 1·13-1·34; p<0·0001) and non-relapse mortality (cause-specific HR for a two-fold change of 1·24, 1·12-1·38; p<0·0001). Model validation for prediction of overall survival and non-relapse mortality by EASIX-GVHD was successful in two independent cohorts of adult patients with reduced-intensity conditioning (n=141, n=173) and in a cohort with mainly paediatric patients (n=89).
INTERPRETATION: In patients with reduced-intensity conditioning, EASIX-GVHD is a powerful predictor of survival after GVHD. EASIX-GVHD could be the future basis for development of risk-adapted GVHD treatment strategies.
FUNDING: There was no external funding source for this study.

PMID: 28733186 [PubMed – as supplied by publisher]

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Infectious and Noninfectious Pulmonary Complications in Patients With Primary Immunodeficiency Disorders.

J Investig Allergol Clin Immunol. 2017;27(4):213-224

Authors: Yazdani R, Abolhassani H, Asgardoon M, Shaghaghi M, Modaresi M, Azizi G, Aghamohammadi A

Abstract
Primary immunodeficiency disorders (PIDs) are caused by 1 or more defects of the immune system. Patients are more likely to experience recurrent and/or severe infections and tend to develop a wide range of complications. Respiratory diseases are the main and initial manifestation in most cases and the most common complication. Pulmonary complications cause significant morbidity and mortality in patients with PIDs. Early diagnosis and appropriate treatment can prevent or at least slow the development of respiratory complications. Since the spectrum of pulmonary complications in PIDs is broad, we divided pulmonary complications into upper respiratory complications (eg, sinusitis, otitis media, and laryngeal angioedema) and lower respiratory complications (eg, pneumonia, bronchitis, bronchiectasis, interstitial lung diseases, organizing pneumonia, pulmonary adenopathies and malignancies, hyperreactive airway diseases, pulmonary dysgenesis, and adverse reactions to treatment). This review covers the main respiratory manifestations in patients with PIDs.

PMID: 28731410 [PubMed – in process]

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