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Article ; Online: Diagnostic Testing for Chronic Granulomatous Disease.

Kuhns, Douglas B

Methods in molecular biology (Clifton, N.J.)

2019 Volume 1982, Page(s) 543–571

Abstract: Chronic granulomatous disease (CGD) is a rare genetic immunodeficiency associated with recurrent bacterial infections, granulomas, and increased mortality. It is characterized by the inability of phagocytes (neutrophils, monocytes, etc.) to generate ... ...

Abstract	Chronic granulomatous disease (CGD) is a rare genetic immunodeficiency associated with recurrent bacterial infections, granulomas, and increased mortality. It is characterized by the inability of phagocytes (neutrophils, monocytes, etc.) to generate reactive oxygen species (ROS), a major component of the microbicidal repertoire of phagocytes. Diagnosis of patients with CGD is commonly based on the assessment of ROS production by neutrophils. Multiple assays to assess ROS production are described-a flow cytometric dihydrorhodamine assay and a histochemical nitroblue tetrazolium assay, both of which can be used to visualize ROS production in individual cells, and two quantitative assays-O
MeSH term(s)	Biological Assay ; Biomarkers ; Flow Cytometry ; Granulomatous Disease, Chronic/diagnosis ; Granulomatous Disease, Chronic/etiology ; Granulomatous Disease, Chronic/metabolism ; Humans ; Molecular Diagnostic Techniques ; NADPH Oxidases/chemistry ; NADPH Oxidases/genetics ; NADPH Oxidases/metabolism ; Neutrophils/immunology ; Neutrophils/metabolism ; Phagocytes/immunology ; Phagocytes/metabolism ; Reactive Oxygen Species/metabolism
Chemical Substances	Biomarkers ; Reactive Oxygen Species ; NADPH Oxidases (EC 1.6.3.-)
Language	English
Publishing date	2019-06-06
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-9424-3_33
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Article ; Online: A Heterozygous Gain-of-Function Variant in IKBKB Associated with Autoimmunity and Autoinflammation.

Sacco, Keith / Kuehn, Hye Sun / Kawai, Tomoki / Alsaati, Nouf / Smith, Lauren / Davila, Blachy / Bundy, Vanessa / Kuhns, Douglas B / Dobbs, Kerry / Delmonte, Ottavia / Notarangelo, Luigi D / Rosenzweig, Sergio D / Keller, Michael D

Journal of clinical immunology

2022 Volume 43, Issue 2, Page(s) 512–520

Abstract: ... Jurkat cells transduced with the IKKb T559M variant showed increased basal levels of phosphorylation of IKKα/b ...

Abstract	Purpose: Biallelic loss-of-function variants in IKBKB cause severe combined immunodeficiency. We describe a case of autoimmunity and autoinflammation in a male infant with a heterozygous gain-of-function (GOF) IKBKB variant. Methods: Case report and review of the literature. We performed in silico variant analysis, measurement of plasma soluble biomarkers associated with immune activation, functional stimulation of patient peripheral blood mononuclear cells, and functional validation of variants transduced in Jurkat cells. Results: A patient with two heterozygous IKBKB variants (E518K and T559M) presents with previously undescribed autoimmune cytopenias and autoinflammation. He had decreased TNF-α-induced IkBα degradation in vitro, and had increased serum biomarkers associated with macrophage recruitment and activation. Jurkat cells transduced with the IKKb T559M variant showed increased basal levels of phosphorylation of IKKα/b and p65, and higher degradation of IkBα suggesting a GOF mechanism. No significant changes were observed in Jurkat cells transduced with the E518K variant. Conclusions: A GOF variant in IKBKB may associate with autoinflammation and autoimmunity highlighting a novel clinical phenotype.
MeSH term(s)	Male ; Humans ; Autoimmunity/genetics ; I-kappa B Kinase/genetics ; Gain of Function Mutation ; Leukocytes, Mononuclear ; Biomarkers
Chemical Substances	I-kappa B Kinase (EC 2.7.11.10) ; Biomarkers ; IKBKB protein, human (EC 2.7.11.10)
Language	English
Publishing date	2022-11-15
Publishing country	Netherlands
Document type	Review ; Case Reports ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	779361-3
ISSN	1573-2592 ; 0271-9142
ISSN (online)	1573-2592
ISSN	0271-9142
DOI	10.1007/s10875-022-01395-2
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Article ; Online: Interpretation of Dihydrorhodamine-1,2,3 Flow Cytometry in Chronic Granulomatous Disease: an Atypical Exemplar.

Donko, Agnes / Kuhns, Douglas B / Cousin, Margot A / Smith, Matthew J / Sacco, Keith A / Klee, Eric W / Joshi, Avni Y / Gavrilova, Ralitza H / Holland, Steven M / Leto, Thomas L / Abraham, Roshini S

Journal of clinical immunology

2022 Volume 42, Issue 5, Page(s) 986–999

Abstract: Purpose: This is a functional characterization of a novel CYBA variant associated with normal DHR flow cytometry. Chronic granulomatous disease (CGD) is an inborn error of immunity characterized by recurrent bacterial and fungal infections and ... ...

Abstract	Purpose: This is a functional characterization of a novel CYBA variant associated with normal DHR flow cytometry. Chronic granulomatous disease (CGD) is an inborn error of immunity characterized by recurrent bacterial and fungal infections and dysregulated inflammatory responses due to defective phagocytic cell function leading to the formation of granulomas. CGD patients have pathogenic variants in any of the five components of the phagocytic NADPH oxidase, which transfers electrons through the phagosomal membrane and produces superoxide upon bacterial uptake. Here, we report a pediatric female patient with a novel homozygous missense variant (c.293C > T, p.(Ser98Leu)) in CYBA, encoding the p22 Methods and results: The patient presented with severe recurrent pneumonia. Specific pathogens identified included Burkholderia and Serratia species suggesting neutrophil functional abnormalities; however, the dihydrorhodamine-1,2,3 (DHR) flow cytometric and cytochrome c reduction assays for neutrophil respiratory burst fell within the low side of the normal range. Western blot and flow cytometric analysis of individual NADPH oxidase components revealed reduced levels of p22 Conclusions: Although this patient did not exhibit all the classic features of CGD, such as granulomas and skin infections, she had recurrent pneumonias with oxidant-sensitive pathognomonic organisms, resulting in appropriate targeted CGD testing. This case emphasizes the need to contextually interpret laboratory data, especially using clinical findings to direct additional assessments including genetic analysis.
MeSH term(s)	Child ; Female ; Flow Cytometry ; Granulomatous Disease, Chronic/complications ; Granulomatous Disease, Chronic/diagnosis ; Granulomatous Disease, Chronic/genetics ; Humans ; Mutation/genetics ; NADPH Oxidase 2/genetics ; NADPH Oxidases/genetics ; Phagocytes
Chemical Substances	NADPH Oxidase 2 (EC 1.6.3.-) ; NADPH Oxidases (EC 1.6.3.-) ; CYBA protein, human (EC 1.6.3.1)
Language	English
Publishing date	2022-03-28
Publishing country	Netherlands
Document type	Case Reports ; Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	779361-3
ISSN	1573-2592 ; 0271-9142
ISSN (online)	1573-2592
ISSN	0271-9142
DOI	10.1007/s10875-022-01217-5
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Article ; Online: Hematologically important mutations: Leukocyte adhesion deficiency (second update).

Blood cells, molecules & diseases

2023 Volume 99, Page(s) 102726

Abstract: Leukocyte adhesion deficiency (LAD) is an immunodeficiency caused by defects in the adhesion of leukocytes (especially neutrophils) to the blood vessel wall. As a result, patients with LAD suffer from severe bacterial infections and impaired wound ... ...

Abstract	Leukocyte adhesion deficiency (LAD) is an immunodeficiency caused by defects in the adhesion of leukocytes (especially neutrophils) to the blood vessel wall. As a result, patients with LAD suffer from severe bacterial infections and impaired wound healing, accompanied by neutrophilia. In LAD-I, characterized directly after birth by delayed separation of the umbilical cord, mutations are found in ITGB2, the gene that encodes the β subunit (CD18) of the β
MeSH term(s)	Humans ; Cell Adhesion/genetics ; Leukocyte-Adhesion Deficiency Syndrome/genetics ; CD18 Antigens/genetics ; CD18 Antigens/metabolism ; Leukocytes ; Mutation
Chemical Substances	CD18 Antigens
Language	English
Publishing date	2023-01-20
Publishing country	United States
Document type	Journal Article
ZDB-ID	1237083-6
ISSN	1096-0961 ; 1079-9796
ISSN (online)	1096-0961
ISSN	1079-9796
DOI	10.1016/j.bcmd.2023.102726
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Article ; Online: Editorial: will the real neutrophil please stand up?

Zarember, Kol A / Kuhns, Douglas B

Journal of leukocyte biology

2011 Volume 90, Issue 6, Page(s) 1039–1041

MeSH term(s)	Animals ; Cell Communication/immunology ; Endothelium, Vascular/cytology ; Endothelium, Vascular/immunology ; Endothelium, Vascular/pathology ; Humans ; Neutrophils/cytology ; Neutrophils/immunology ; Neutrophils/pathology ; Stem Cells/cytology ; Stem Cells/immunology ; Stem Cells/pathology ; Transendothelial and Transepithelial Migration/immunology
Language	English
Publishing date	2011-11-30
Publishing country	United States
Document type	Editorial ; Research Support, N.I.H., Extramural
ZDB-ID	605722-6
ISSN	1938-3673 ; 0741-5400
ISSN (online)	1938-3673
ISSN	0741-5400
DOI	10.1189/jlb.0711334
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Article ; Online: A phase III randomized crossover trial of plerixafor versus G-CSF for treatment of WHIM syndrome.

The Journal of clinical investigation

2023 Volume 133, Issue 19

Abstract: BACKGROUNDWarts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a primary immunodeficiency disorder caused by heterozygous gain-of-function CXCR4 mutations. Myelokathexis is a kind of neutropenia caused by neutrophil retention in ...

Abstract	BACKGROUNDWarts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome is a primary immunodeficiency disorder caused by heterozygous gain-of-function CXCR4 mutations. Myelokathexis is a kind of neutropenia caused by neutrophil retention in bone marrow and in WHIM syndrome is associated with lymphopenia and monocytopenia. The CXCR4 antagonist plerixafor mobilizes leukocytes to the blood; however, its safety and efficacy in WHIM syndrome are undefined.METHODSIn this investigator-initiated, single-center, quadruple-masked phase III crossover trial, we compared the total infection severity score (TISS) as the primary endpoint in an intent-to-treat manner in 19 patients with WHIM who each received 12 months treatment with plerixafor and 12 months treatment with granulocyte CSF (G-CSF, the standard of care for severe congenital neutropenia). The treatment order was randomized for each patient.RESULTSPlerixafor was nonsuperior to G-CSF for TISS (P = 0.54). In exploratory endpoints, plerixafor was noninferior to G-CSF for maintaining neutrophil counts of more than 500 cells/μL (P = 0.023) and was superior to G-CSF for maintaining lymphocyte counts above 1,000 cells/μL (P < 0.0001). Complete regression of a subset of large wart areas occurred on plerixafor in 5 of 7 patients with major wart burdens at baseline. Transient rash occurred on plerixafor, and bone pain was more common on G-CSF. There were no significant differences in drug preference or quality of life or the incidence of drug failure or serious adverse events.CONCLUSIONPlerixafor was not superior to G-CSF in patients with WHIM for TISS, the primary endpoint. Together with wart regression and hematologic improvement, the infection severity results support continued study of plerixafor as a potential treatment for WHIM syndrome.TRIAL REGISTRATIONClinicaltrials.gov NCT02231879.FUNDINGThis study was funded by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases.
MeSH term(s)	Humans ; Immunologic Deficiency Syndromes/drug therapy ; Immunologic Deficiency Syndromes/genetics ; Granulocyte Colony-Stimulating Factor/therapeutic use ; Hematopoietic Stem Cell Mobilization/adverse effects ; Cross-Over Studies ; Quality of Life ; Heterocyclic Compounds/adverse effects ; Primary Immunodeficiency Diseases/drug therapy ; Primary Immunodeficiency Diseases/genetics ; Warts/drug therapy ; Warts/genetics ; Receptors, CXCR4/genetics
Chemical Substances	Granulocyte Colony-Stimulating Factor (143011-72-7) ; Heterocyclic Compounds ; Receptors, CXCR4
Language	English
Publishing date	2023-10-02
Publishing country	United States
Document type	Randomized Controlled Trial ; Clinical Trial, Phase III ; Journal Article ; Research Support, N.I.H., Intramural ; Research Support, N.I.H., Extramural
ZDB-ID	3067-3
ISSN	1558-8238 ; 0021-9738
ISSN (online)	1558-8238
ISSN	0021-9738
DOI	10.1172/JCI164918
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Article ; Online: Anakinra-Associated Systemic Amyloidosis.

Arthritis & rheumatology (Hoboken, N.J.)

2023 Volume 76, Issue 1, Page(s) 100–106

Abstract: Objective: To describe a 41-year-old woman with a history of neonatal onset multisystem inflammatory disease, on treatment with daily subcutaneous injections of 600 mg of recombinant interleukin-1 receptor antagonist (IL-1Ra) protein, anakinra, since ... ...

Abstract	Objective: To describe a 41-year-old woman with a history of neonatal onset multisystem inflammatory disease, on treatment with daily subcutaneous injections of 600 mg of recombinant interleukin-1 receptor antagonist (IL-1Ra) protein, anakinra, since the age of 28, who presented with golf-ball size nodules at the anakinra injection sites, early satiety, new onset nephrotic syndrome in the context of normal markers of systemic inflammation. Methods: Clinical history and histologic evaluation of biopsies of skin, gastric mucosa, and kidney with Congo-red staining and proteomic evaluation of microdissected Congo red-positive amyloid deposits by liquid chromatography-tandem mass spectrometry. Results: The skin, stomach, and kidney biopsies all showed the presence of Congo red-positive amyloid deposits. Mass spectrometry-based proteomics demonstrated that the amyloid deposits in all sites were of AIL1RAP (IL-1Ra protein)-type. These were characterized by high spectral counts of the amyloid signature proteins (apolipoprotein AIV, apolipoprotein E, and serum amyloid P-component) and the amyloidogenic IL-1Ra protein, which were present in Congo red-positive areas and absent in Congo red-negative areas. The amino acid sequence identified by mass spectrometry confirmed that the amyloid precursor protein was recombinant IL-1Ra (anakinra) and not endogenous wild-type IL-1Ra. Conclusion: This is the first report of iatrogenic systemic amyloidosis due to an injectable protein drug, which was caused by recombinant IL1Ra (anakinra).
MeSH term(s)	Female ; Infant, Newborn ; Humans ; Adult ; Interleukin 1 Receptor Antagonist Protein/therapeutic use ; Plaque, Amyloid ; Congo Red/chemistry ; Proteomics ; Amyloidosis/metabolism ; Amyloidosis/pathology
Chemical Substances	Interleukin 1 Receptor Antagonist Protein ; Congo Red (3U05FHG59S)
Language	English
Publishing date	2023-11-29
Publishing country	United States
Document type	Case Reports ; Journal Article ; Research Support, N.I.H., Intramural
ZDB-ID	2756371-6
ISSN	2326-5205 ; 2326-5191
ISSN (online)	2326-5205
ISSN	2326-5191
DOI	10.1002/art.42664
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Article ; Online: Homozygous variant p. Arg90His in NCF1 is associated with early-onset Interferonopathy: a case report.

Schnappauf, Oskar / Heale, Liane / Dissanayake, Dilan / Tsai, Wanxia L / Gadina, Massimo / Leto, Thomas L / Kastner, Daniel L / Malech, Harry L / Kuhns, Douglas B / Aksentijevich, Ivona / Laxer, Ronald M

Pediatric rheumatology online journal

2021 Volume 19, Issue 1, Page(s) 54

Abstract: Background: Biallelic loss-of-function variants in NCF1 lead to reactive oxygen species deficiency and chronic granulomatous disease (CGD). Heterozygosity for the p.Arg90His variant in NCF1 has been associated with susceptibility to systemic lupus ... ...

Abstract	Background: Biallelic loss-of-function variants in NCF1 lead to reactive oxygen species deficiency and chronic granulomatous disease (CGD). Heterozygosity for the p.Arg90His variant in NCF1 has been associated with susceptibility to systemic lupus erythematosus, rheumatoid arthritis, and Sjögren's syndrome in adult patients. This study demonstrates the association of the homozygous p.Arg90His variant with interferonopathy with features of autoinflammation and autoimmunity in a pediatric patient. Case presentation: A 5-year old female of Indian ancestry with early-onset recurrent fever and headache, and persistently elevated antinuclear, anti-Ro, and anti-La antibodies was found to carry the homozygous p.Arg90His variant in NCF1 through exome sequencing. Her unaffected parents and three other siblings were carriers for the mutant allele. Because the presence of two NCF1 pseudogenes, this variant was confirmed by independent genotyping methods. Her intracellular neutrophil oxidative burst and NCF1 expression levels were normal, and no clinical features of CGD were apparent. Gene expression analysis in peripheral blood detected an interferon gene expression signature, which was further supported by cytokine analyses of supernatants of cultured patient's cells. These findings suggested that her inflammatory disease is at least in part mediated by type I interferons. While her fever episodes responded well to systemic steroids, treatment with the JAK inhibitor tofacitinib resulted in decreased serum ferritin levels and reduced frequency of fevers. Conclusion: Homozygosity for p.Arg90His in NCF1 should be considered contributory in young patients with an atypical systemic inflammatory antecedent phenotype that may evolve into autoimmunity later in life. The complex genomic organization of NCF1 poses a difficulty for high-throughput genotyping techniques and variants in this gene should be carefully evaluated when using the next generation and Sanger sequencing technologies. The p.Arg90His variant is found at a variable allele frequency in different populations, and is higher in people of South East Asian ancestry. In complex genetic diseases such as SLE, other rare and common susceptibility alleles might be necessary for the full disease expressivity.
MeSH term(s)	Autoimmune Diseases/genetics ; Child, Preschool ; Female ; Homozygote ; Humans ; Interferons ; NADPH Oxidases/genetics ; Pedigree
Chemical Substances	Interferons (9008-11-1) ; NADPH Oxidases (EC 1.6.3.-) ; neutrophil cytosolic factor 1 (EC 1.6.3.1)
Language	English
Publishing date	2021-04-23
Publishing country	England
Document type	Case Reports ; Journal Article
ZDB-ID	2279468-2
ISSN	1546-0096 ; 1546-0096
ISSN (online)	1546-0096
ISSN	1546-0096
DOI	10.1186/s12969-021-00536-y
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Article ; Online: Neutrophil activation in systemic capillary leak syndrome (Clarkson disease).

Xie, Zhihui / Kuhns, Douglas B / Gu, Xuesong / Otu, Hasan H / Libermann, Towia A / Gallin, John I / Parikh, Samir M / Druey, Kirk M

Journal of cellular and molecular medicine

2019 Volume 23, Issue 8, Page(s) 5119–5127

Abstract: Systemic capillary leak syndrome (SCLS; Clarkson disease) is a rare orphan disorder characterized by transient yet recurrent episodes of hypotension and peripheral oedema due to diffuse vascular leakage of fluids and proteins into soft tissues. Humoral ... ...

Abstract	Systemic capillary leak syndrome (SCLS; Clarkson disease) is a rare orphan disorder characterized by transient yet recurrent episodes of hypotension and peripheral oedema due to diffuse vascular leakage of fluids and proteins into soft tissues. Humoral mediators, cellular responses and genetic features accounting for the clinical phenotype of SCLS are virtually unknown. Here, we searched for factors altered in acute SCLS plasma relative to matched convalescent samples using multiplexed aptamer-based proteomic screening. Relative amounts of 612 proteins were changed greater than twofold and 81 proteins were changed at least threefold. Among the most enriched proteins in acute SCLS plasma were neutrophil granule components including bactericidal permeability inducing protein, myeloperoxidase and matrix metalloproteinase 8. Neutrophils isolated from blood of subjects with SCLS or healthy controls responded similarly to routine pro-inflammatory mediators. However, acute SCLS sera activated neutrophils relative to remission sera. Activated neutrophil supernatants increased permeability of endothelial cells from both controls and SCLS subjects equivalently. Our results suggest systemic neutrophil degranulation during SCLS acute flares, which may contribute to the clinical manifestations of acute vascular leak.
MeSH term(s)	Adult ; Blood Proteins/genetics ; Capillary Leak Syndrome/blood ; Capillary Leak Syndrome/genetics ; Capillary Leak Syndrome/pathology ; Endothelial Cells ; Endothelium, Vascular/metabolism ; Female ; Humans ; Male ; Middle Aged ; Neutrophil Activation/genetics ; Neutrophils/metabolism ; Proteomics
Chemical Substances	Blood Proteins
Language	English
Publishing date	2019-06-18
Publishing country	England
Document type	Journal Article ; Research Support, N.I.H., Intramural
ZDB-ID	2074559-X
ISSN	1582-4934 ; 1582-4934 ; 1582-1838
ISSN (online)	1582-4934
ISSN	1582-4934 ; 1582-1838
DOI	10.1111/jcmm.14381
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Article ; Online: Isolation and Functional Analysis of Human Neutrophils.

Kuhns, Douglas B / Priel, Debra A Long / Chu, Jessica / Zarember, Kol A

Current protocols in immunology

2015 Volume 111, Page(s) 7.23.1–7.23.16

Abstract: This unit describes the isolation of human polymorphonuclear neutrophils (PMN) from blood using dextran sedimentation and Percoll or Ficoll-Paque density gradients. Assays of neutrophil functions including respiratory burst activation, phagocytosis, and ... ...

Abstract	This unit describes the isolation of human polymorphonuclear neutrophils (PMN) from blood using dextran sedimentation and Percoll or Ficoll-Paque density gradients. Assays of neutrophil functions including respiratory burst activation, phagocytosis, and microbial killing are also described.
MeSH term(s)	Centrifugation, Density Gradient/methods ; Cytochromes c/metabolism ; Dextrans ; Ficoll ; Flow Cytometry/methods ; Humans ; Hydrogen Peroxide/analysis ; Hydrogen Peroxide/metabolism ; Neutrophils/cytology ; Neutrophils/immunology ; Phagocytosis/immunology ; Povidone ; Respiratory Burst/physiology ; Silicon Dioxide ; Staphylococcus aureus/immunology ; Superoxide Dismutase/metabolism
Chemical Substances	Dextrans ; Ficoll (25702-74-3) ; Percoll (65455-52-9) ; Silicon Dioxide (7631-86-9) ; Cytochromes c (9007-43-6) ; Hydrogen Peroxide (BBX060AN9V) ; Superoxide Dismutase (EC 1.15.1.1) ; Povidone (FZ989GH94E)
Language	English
Publishing date	2015-11-02
Publishing country	United States
Document type	Journal Article ; Research Support, N.I.H., Extramural ; Research Support, N.I.H., Intramural
ZDB-ID	2179059-0
ISSN	1934-368X ; 1934-3671
ISSN (online)	1934-368X
ISSN	1934-3671
DOI	10.1002/0471142735.im0723s111
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