Dihydrorhodamine Flow Cytometry Research Articles

Chronic granulomatous disease with an initial presentation of arthritis and oral ulcers

Chronic granulomatous disease (CGD) is an inherited defect of leukocyte phagocytic function leading to recurrent infections. Autoimmune manifestations are reported in up to 6% of patients with CGD. We report a case of CGD presenting with arthritis as the first manifestation of disease. A 12-year-old Pakistani male of consanguineous parents presented with migratory arthritis and painless oral ulcerations of 6 months duration that were minimally responsive to nonsteroidal anti-inflammatory treatment. Initial assessment demonstrated elevated inflammatory markers (ESR 62), weakly positive ANA (titer 1:40), negative anti-DsDNA, and negative RF. He presented to the emergency department with fevers and arthritis. Repeat work-up suggested early Macrophage Activation Syndrome: normocytic anemia (Hgb 95 g/L), thrombocytopenia (Plt 141 × 109/L), elevated LDH 1603, ferritin 1230 mcg/L, ESR 127, CRP 9.3, hypertiglycerdemia (3.2 mmoL/L) and mild transaminitis (ALT 63, AST 87), normal bone marrow (no hemophagocytosis), but mildly elevated Soluble CD 136 (1086 ng/mL) and Soluble IL-2 receptor (CD25) (1698 U/mL). He was treated with oral prednisone with symptom resolution. The arthritis relapsed after 1 month and the patient developed fever, productive cough, and pleuritic chest pain. Chest imaging revealed multiple nodular opacities and enlarged mediastinal lymph nodes. Aspergillus fumigatus complex was isolated from induced sputum prompting screening for primary immunodeficiency. Neutrophil oxidative burst function, as assessed by a dihydrorhodamine flow cytometry based assay, was low at 1.26 and 1.48 (normal range 32–300). Genetic analysis showed a previously described mutation in the NCF1 gene confirming the diagnosis of autosomal-recessive CGD. CGD can present with an exclusively rheumatologic presentation including arthritis and oral ulceration. Statement of Novelty: This case demonstrates that CGD can present with rheumatological symptoms prior to any infectious features.

332. ZFN-Mediated Minigene or Dinucleotide Gene Correction of p47phox Deficient Autosomal Recessive Chronic Granulomatous Disease iPSC to Generate Oxidase Functional Neutrophils

Chronic granulomatous disease is an immune deficiency caused by defective phagocyte NADPH oxidase resulting in severe infections and autoimmune complications. NCF1 gene mutations resulting in p47phox deficiency are the most common cause of autosomal-recessive chronic granulomatous disease (AR-CGD), and notably, >80% of patients have homozygous GT-deletions in exon 2, resulting in a frame shift, premature termination, and absence of p47phox expression. Here we show two nucleofection strategies for NCF1 gene correction using zinc-finger nuclease (ZFN) targeting the NCF1 locus in patient EBV-immortalized B cells and in induced pluripotent stem cells (iPSC) derived from p47phox-deficient patients. With the first strategy, ZFN-mediated cut at start of exon 2 allows insertion of a minigene (NCF1 cDNA with deleted exon 1) to allow continuous transcription from the endogenous NCF1 exon 1 to the donor minigene exon 2 until the end of the cDNA and beta-globin polyadenylation signal. The second strategy takes advantage of the most prevalent GT-deletion by providing a dinucleotide corrective donor plasmid for gene editing using the same ZFNs. The two donors contain ~750 bp to 1000 bp flanking homology arms and also encode a selectable marker: a puromycin resistance gene flanked by loxP sites to allow for excision after selection. In patient EBV-immortalized B cells, minigene and dinucleotide targeted gene correction of constitutively active NCF1 was 8-20% after puromycin-selection from obtained clones. The rates of correction in iPSC were generally lower, less than 2%. Use of AAV2 vector to deliver dinucleotide corrective donor to iPSC increased insertion efficiency to >80% after puromycin selection. Gene corrected B cells for both correction strategies restored ROS function, shown by chemiluminescence assay. Neutrophils differentiated from gene corrected iPSCs demonstrated correction of oxidase activity by chemiluminescence or dihydrorhodamine flow cytometry assay. With both methods, p47phox protein expression is seen in neutrophils differentiated from iPSC but not in undifferentiated iPSC, demonstrating cell type-specific regulation of expression at the corrected locus. In summary, we show that iPSC generated from CGD patient cells can be gene corrected through a targeted dinucleotide correction method for greater than 80% of alleles and minigene correction for mutations other than the GT-deletions.

Light emitting diode-generated blue light modulates fibrosis characteristics: fibroblast proliferation, migration speed, and reactive oxygen species generation.

Blue light is part of the visible light spectrum that does not generate harmful DNA adducts associated with skin cancer and photoaging, and may represent a safer therapeutic modality for treatment of keloid scars and other fibrotic skin diseases. Our laboratory previously demonstrated that light-emitting diode (LED) red and infrared light inhibits proliferation of skin fibroblasts. Moreover, different wavelengths of light can produce different biological effects. Furthermore, the effects of LED blue light (LED-BL) on human skin fibroblasts are not well characterized. This study investigated the effects of LED-BL on human skin fibroblast proliferation, viability, migration speed, and reactive oxygen-species (ROS) generation. Irradiation of adult human skin fibroblasts using commercially-available LED-BL panels was performed in vitro, and modulation of proliferation and viability was quantified using the trypan blue dye exclusion assay, migratory speed was assessed using time-lapse video microscopy, and intracellular ROS generation was measured using the dihydrorhodamine flow cytometry assay. Statistical differences between groups were determined by ANOVA and Student's t-test. Human skin fibroblasts treated with LED-BL fluences of 5, 10, 15, 30, and 80 J/cm(2) demonstrated statistically significant dose-dependent decreases in relative proliferation of 8.4%, 29.1%, 33.8%, 51.7%, and 55.1%, respectively, compared to temperature and environment matched bench control plates, respectively. LED-BL fluences of 5, 30, 45, and 80 J/cm(2) decreased fibroblast migration speed to 95 ± 7.0% (P = 0.64), 81.3 ± 5.5% (P = 0.021), 48.5 ± 2.7% (P < 0.0001), and 32.3 ± 1.9% (P < 0.0001), respectively, relative to matched controls. LED fluences of 5, 10, 30, and 80 J/cm(2) resulted in statistically significant increases in reactive oxygen species of 110.4%, 116.6%, 127.5%, and 130%, respectively, relative to bench controls. At the fluences studied, LED-BL can inhibit adult human skin dermal fibroblast proliferation and migration speed, and is associated with increased reactive oxygen species generation in a dose-dependent manner without altering viability. LED-BL has the potential to contribute to the treatment of keloids and other fibrotic skin diseases and is worthy of further translational and clinical investigation.

Dual stimulus-dependent effect of Oenothera paradoxa extract on the respiratory burst in human leukocytes: suppressing for Escherichia coli and phorbol myristate acetate and stimulating for formyl-methionyl-leucyl-phenylalanine.

Although a growing body of evidence suggests that plant polyphenols can modulate human immune responses, their simultaneous action on monocyte and neutrophil oxidative burst is currently poorly understood. Based on the hypothesis that various polyphenols contained in plant extracts might affect the oxidative burst of phagocytes, we evaluated the effects of ethanolic O. paradoxa extract polyphenols on monocyte and neutrophil oxidative burst in vitro activated by different stimuli, including opsonized bacteria E. coli, phorbol 12-myristate 13-acetate (PMA), and formyl-methionyl-leucyl-phenylalanine (fMLP). Samples were analyzed by the dihydrorhodamine flow cytometry assay. Our results showed that the extract repressed significantly and dose-dependently reactive oxygen species production in both cell types stimulated with E. coli and PMA (P < 0.05) and its inhibitory efficiency was stimulus- and cell-type-dependent. Interestingly, there was significant stimulatory effect of the extract on bursting phagocytes induced by fMLP (P < 0.05). Additionally, several flavonoids and phenolic compounds as well as penta-galloyl-β-(D)-glucose (PGG), the representative of hydrolyzable tannins, were identified in the 60% extract by high-performance liquid chromatography (HPLC) coupled to electrospray ionization in negative ion mode. In summary, the ethanolic O. paradoxa extract, rich in flavonoids and phenolic compounds, exhibits dual stimulus-dependent effect on the respiratory burst in human leukocytes; hence, it might affect immune responses in humans.

Chemiluminescent and flow cytometric analysis of gamma interferon preincubation on neonatal and adult rat polymorphonuclear leukocytes.

Gamma interferon (IFN-gamma) has multiple immunomodulating effects and has been postulated as a possible immunopotentiating agent for the prevention or treatment of neonatal infections. This report describes the effect of rat recombinant IFN-gamma on the oxidative burst activity and CD11b expression of neonatal and adult rat polymorphonuclear leukocytes (PMNL). Oxidative burst activity was assessed by chemiluminescence and dihydrorhodamine flow cytometry. Neonatal PMNL exhibited significantly less oxidative burst activity than did adult PMNL. IFN-gamma mildly enhanced the chemiluminescence response of PMNL from both the rat pups and adults, but this effect was not statistically significant when analyzed by a multivariate model of repeated-measures analysis of variance for both chemiluminescence and dihydrorhodamine flow cytometry. CD11b expression was also not significantly enhanced by IFN-gamma.