Frequency of <i>APOL1</i> G1 and <i>APOL1</i> G2 risk variants for CKD-AFRiKA study sample and other populations of interest.

<p>Frequency of <i>APOL1</i> G1 and <i>APOL1</i> G2 risk variants for CKD-AFRiKA study sample and other populations of interest.</p

Reported frequencies of <i>APOL1</i> G1 and G2 risk alleles for several Bantu populations across sub-Saharan Africa.

<p>Reported frequencies of <i>APOL1</i> G1 and G2 risk alleles for several Bantu populations across sub-Saharan Africa.</p

Characteristics of participants with CKD, by genotyping status; n = 57.

<p>Characteristics of participants with CKD, by genotyping status; n = 57.</p

<i>APOL1</i> risk alleles among individuals with CKD in Northern Tanzania: A pilot study

<div><p>Introduction</p><p>In sub-Saharan Africa, approximately 100 million people have CKD, yet genetic risk factors are not well-understood. Despite the potential importance of understanding <i>APOL1</i> risk allele status among individuals with CKD, little genetic research has been conducted. Therefore, we conducted a pilot study evaluating the feasibility of and willingness to participate in genetic research on kidney disease, and we estimated <i>APOL1</i> risk allele frequencies among individuals with CKD.</p><p>Methods</p><p>In 2014, we conducted a community-based field study evaluating CKD epidemiology in northern Tanzania. We assessed for CKD using urine albumin and serum creatinine to estimate GFR. We invited participants with CKD to enroll in an additional genetic study. We obtained dried-blood spots on filter cards, from which we extracted DNA using sterile punch biopsies. We genotyped for two single nucleotide polymorphisms (SNPs) defining the <i>APOL1</i> G1 risk allele and an insertion/deletion polymorphism defining the G2 risk allele. Genotyping was performed in duplicate.</p><p>Results</p><p>We enrolled 481 participant, 57 (12%) of whom had CKD. Among these, enrollment for genotyping was high (n = 48; 84%). We extracted a median of 19.4 ng of DNA from each dried-blood spot sample, and we genotyped the two <i>APOL1</i> G1 SNPs and the <i>APOL1</i> G2 polymorphism. Genotyping quality was high, with all duplicated samples showing perfect concordance. The frequency of <i>APOL1</i> risk variants ranged from 7.0% to 11.0%, which was similar to previously-reported frequencies from the general population of northern Tanzania (p>0.2).</p><p>Discussion</p><p>In individuals with CKD from northern Tanzania, we demonstrated feasibility of genotyping <i>APOL1</i> risk alleles. We successfully genotyped three risk variants from DNA extracted from filter cards, and we demonstrated a high enrollment for participation. In this population, more extensive genetic studies of kidney disease may be well-received and will be feasible.</p></div

Analysis of polymorphisms in a United States population with pseudoexfoliation glaucoma-0

<p><b>Copyright information:</b></p><p>Taken from "Analysis of polymorphisms in a United States population with pseudoexfoliation glaucoma"</p><p></p><p>Molecular Vision 2008;14():146-149.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2255060.</p><p></p

Intra-bronchial epithelial Shp2 depletion exert little effects on OVA-induced inflammation and TH2 and TH17 polarization.

<p>Twenty-four hours after the last challenge, the cellular profiles (A) were analyzed, and total cell numbers (B) were counted in BALF of mice. (C) IL-4, IL-17 and foxp3 mRNA were measured in the lung tissues of the mice. (D) Ratios of TH2 (stained by FITC-CD4 and APC-IL-4) and TH17 (stained by FITC-CD4 and PE-IL-17A) in the lung homogenates. (n = 5 mice/group, in two separate experiments). Results were expressed as mean ± SEM. ^n.s.<i>p</i> >0.05. PBS_ave: average value of three PBS subgroups, including <i>CC10-rtTA /Shp2</i>^<i>f/f</i>:DOX (toxicity control, TC), <i>CC10-rtTA/(tetO)7-Cre/Shp2</i>^<i>f/f</i>:H₂O (Shp2^F/F), and <i>CC10-rtTA/(tetO)7-Cre/Shp2</i>^<i>f/f</i>:DOX (Shp2^△/△). ^n.s.<i>p>0</i>.<i>05</i>. Eos: Eosinophil, Neu: Neutrophil, Lym: Lymphocyte, Macro: Macrophage.</p

Knockout of <i>Shp2</i> in bronchial epithelial cells have a mild effect on epithelium-derived cytokine production <i>in vivo</i>.

<p>(A) Schematic map of the generation of triple-transgenic mice <i>CC10-rtTA/(tetO)7-Cre/Shp2</i>^<i>f/f</i>. The human <i>CC10</i> promoter was used to express the reverse tetracycline transactivator (<i>rtTA</i>) in clara cells. In the presence of DOX, <i>rtTA</i> binds to the <i>tetO-CMV</i> promoter, activating the transcription of Cre-recombinase, removing the floxed exon 4 of the <i>Shp2</i> gene, resulting in specific deletion of <i>shp2</i> in Clara cells. (B) Genotyping was performed by PCR assays using mouse tail genomic DNA. (C) Inactivation of shp2 allele was confirmed by PCR of genomic DNA isolated from lung tissues of <i>CC10-rtTA/(tetO)7-Cre/Shp2</i>^<i>f/f</i> mice after 7-day treatment with DOX (through drinking, 2 mg/ml in H₂O) or H₂O (as control). (D) OVA-induced allergic asthmatic mouse model. Five-to-seven-week animals were given drinking water with or without DOX (2mg/ml) from Day 0, then they were sensitized with intra-peritoneal injection of OVA (80ug/mice) or PBS on Day 7 and Day 21. Three consecutive challenges of OVA aerosol (1%) once a day for 3 continual days from Day 32 to Day 34 were performed; the mice were then sacrificed. (E) The levels of IL-25 and IL-33 mRNA in lung tissues were detected by RT-PCR. Results were expressed as mean ± SEM of three independent experiments. ^n.s.<i>p</i>>0.05. CF: <i>CC10-rtTA/(tetO)</i>_<i>7</i><i>-Cre/Shp2</i>^<i>f/f</i>, CTF: <i>CC10-rtTA/Shp2</i>^<i>f/f</i>. PBS_ave: average value of three PBS subgroups, including <i>CC10-rtTA /Shp2</i>^<i>f/f</i>:DOX (toxicity control, TC), <i>CC10-rtTA/(tetO)7-Cre/Shp2</i>^<i>f/f</i>:H₂O (Shp2^F/F) and <i>CC10-rtTA/(tetO)7-Cre/Shp2</i>^<i>f/f</i>:DOX (Shp2^△/△).</p

OVA- or LPS-elicited production of IL-25.

<p>(A) C57BL/6 mice were sensitized and challenged with OVA, and IL-25 mRNA in lung tissue was measured by q-PCR. (B) MTECs were treated with 20 mg/ml OVA for different periods of time (0, 8, 24 and 48 hours), followed by detection of IL-25 mRNA level. Beas-2b cells were treated with OVA of different concentrations (2 and 20 mg/ml) for 8 hours (C) or 20 mg/ml OVA for different periods of time (0, 2, 8 hours) (D), followed by detection of IL-25 mRNA. Serum-free Beas-2bs were stimulated by LPS of different concentrations (0 ng/ml, 10 ng/ml, 100 ng/ml). IL-25 mRNA (E) were detected by q-PCR, IL-25 protein released into the supernatant (F) were measured by ELISA. MTECs were treated with 100 ng/ml LPS for different periods of time (0, 8, 24 and 48 hours), IL-25 mRNA (G) were detected by q-PCR, IL-25 protein released into the supernatant (H) were measured by ELISA. Results were expressed as mean ± SEM of three independent experiments. B2Bs: Beas-2b cells. *<i>p</i> <0.05, **<i>p</i><0.01 ***<i>p</i><0.001. B2Bs: Beas-2b cells.</p

Deletion of Shp2 in bronchial epithelial cells impairs IL-25 production <i>in vitro</i>, but has minor influence on asthmatic inflammation <i>in vivo</i>

<div><p>Shp2 played an important role in cigarette-smoke-mediated inflammation, surfactant homeostasis and asthmatic airway remodeling. However, whether shp2 plays a key role in epithelium-associated allergic reaction is still unknown. In this study, LPS and OVA were observed to induce the production of IL-25 in bronchial epithelial cells <i>in vitro</i> via the activation of MAPK p38 and JNK. Furthermore, blockage of Shp2 by its specific inhibitor PHPS1 or by siRNA-mediated depletion was found to reduce the production of IL-25 in epithelial cells as well as the up-regulated LPS-triggered activation of JNK but not p38. To confirm the role of intra-bronchial epithelial Shp2 in OVA-induced allergic reaction, we generated <i>CC10-rtTA/(tetO)7-Cre/Shp2</i>^<i>f/f</i> mice, where <i>Shp2</i> was conditionally knocked out in bronchial epithelial cells. Surprisingly, specific deletion of <i>Shp2</i> in bronchial epithelial cells showed a mild but insignificant effect on the expressions of epithelium-derived cytokines as well as TH2 and TH17 polarization following allergen-induced murine airway inflammation. Collectively, our data suggested that deletion of Shp2 impaired IL-25 production in bronchial epithelial cells <i>in vitro</i>, but might yet have minor influence on OVA-induced allergic reaction <i>in vivo</i>.</p></div

LPS-induced IL-25 via the activation of MAPK p38 and JNK.

<p>(A) Serum-free Beas-2bs were treated with different concentrations (10, 100 and 1000 ng/ml) of LPS for different periods of time (15 and 30 min). Phosphorylation levels of p38 and JNK were measured via immunoblotting. (B) Beas-2bs were pre-treated with SB202190 and/or SP600125 30 min, followed by stimulation of LPS. IL-25 protein concentration of the cell culture supernatant was measured in the cell culture 8 hours after giving LPS. Results were expressed as mean ± SEM of three independent experiments. ***<i>p</i><0.001, ^n.s.<i>p>0</i>.<i>05</i>.</p