Kunzhong Zhang

Tight junction defects in patients with atopic dermatitis.

BACKGROUND Atopic dermatitis (AD) is characterized by dry skin and a hyperactive immune response to allergens, 2 cardinal features that are caused in part by epidermal barrier defects. Tight junctions (TJs) reside immediately below the stratum corneum and regulate the selective permeability of the paracellular pathway. OBJECTIVE We evaluated the expression/function of the TJ protein claudin-1 in epithelium from AD and nonatopic subjects and screened 2 American populations for single nucleotide polymorphisms in the claudin-1 gene (CLDN1). METHODS Expression profiles of nonlesional epithelium from patients with extrinsic AD, nonatopic subjects, and patients with psoriasis were generated using Illuminas BeadChips. Dysregulated intercellular proteins were validated by means of tissue staining and quantitative PCR. Bioelectric properties of epithelium were measured in Ussing chambers. Functional relevance of claudin-1 was assessed by using a knockdown approach in primary human keratinocytes. Twenty-seven haplotype-tagging SNPs in CLDN1 were screened in 2 independent populations with AD. RESULTS We observed strikingly reduced expression of the TJ proteins claudin-1 and claudin-23 only in patients with AD, which were validated at the mRNA and protein levels. Claudin-1 expression inversely correlated with T(H)2 biomarkers. We observed a remarkable impairment of the bioelectric barrier function in AD epidermis. In vitro we confirmed that silencing claudin-1 expression in human keratinocytes diminishes TJ function while enhancing keratinocyte proliferation. Finally, CLDN1 haplotype-tagging SNPs revealed associations with AD in 2 North American populations. CONCLUSION Collectively, these data suggest that an impairment in tight junctions contributes to the barrier dysfunction and immune dysregulation observed in AD subjects and that this may be mediated in part by reductions in claudin-1.

Antioxidant Properties of Quercetin

UNLABELLED Quercetin, a plant-derived aglycone form of flavonoid glycosides, has been used as a nutritional supplement and may be beneficial against a variety of diseases, including cancer. We examined the antioxidant properties of quercetin. The reduction potential of quercetin was measured at various pH values using voltammetric methods, and its total antioxidant capacity (TAC) was measured using the phosphomolybdenum method. The effect of quercetin on production of reactive oxygen species (ROS) and nitric oxide (NO) in LPS-stimulated human THP-1 acute monocytic leukemia cells was determined by flow cytometry using CM-H2DCFDA dye. The results were compared with curcumin, a natural product exhibiting a similar range of reported health benefits. RESULTS 1) Quercetin has a higher reduction potential compared with curcumin at three different pH settings and is comparable to Trolox at pH 7-9.5; 2) its TAC is 3.5 fold higher than curcumin; 3) it reduced LPS-induced ROS to near normal levels; 4) it reduced LPS-induced NO production. These data provide a physico-chemical basis for comparing antioxidants, with potential benefits individually or in combination.

Mitigation Effect of an FGF-2 Peptide on Acute Gastrointestinal Syndrome after High-Dose Ionizing Radiation

PURPOSE Acute gastrointestinal syndrome (AGS) resulting from ionizing radiation causes death within 7 days. Currently, no satisfactory agent exists for mitigation of AGS. A peptide derived from the receptor binding domain of fibroblast growth factor 2 (FGF-P) was synthesized and its mitigation effect on AGS was examined. METHODS AND MATERIALS A subtotal body irradiation (sub-TBI) model was created to induce gastrointestinal (GI) death while avoiding bone marrow death. After 10.5 to 16 Gy sub-TBI, mice received an intramuscular injection of FGF-P (10 mg/kg/day) or saline (0.2 ml/day) for 5 days; survival (frequency and duration) was measured. Crypt cells and their proliferation were assessed by hematoxylin, eosin, and BrdU staining. In addition, GI hemoccult score, stool formation, and plasma levels of endotoxin, insulin, amylase, interleukin (IL)-6, keratinocyte-derived chemokine (KC) monocyte chemoattractant protein 1 (MCP-1) and tumor necrosis factor (TNF)-alpha were evaluated. RESULTS Treatment with FGF-P rescued a significant fraction of four strains of mice (33-50%) exposed to a lethal dose of sub-TBI. Use of FGF-P improved crypt survival and repopulation and partially preserved or restored GI function. Furthermore, whereas sub-TBI increased plasma endotoxin levels and several pro-inflammation cytokines (IL-6, KC, MCP-1, and TNF-alpha), FGF-P reduced these adverse responses. CONCLUSIONS The study data support pursuing FGF-P as a mitigator for AGS.

Replication of Murine Mitochondrial DNA Following Irradiation

The effect of radiation on the mitochondrial genome in vivo is largely unknown. Though mitochondrial DNA (mtDNA) is vital for cellular survival and proliferation, it has little DNA repair machinery compared with nuclear DNA (nDNA). A better understanding of how radiation affects mtDNA should lead to new approaches for radiation protection. We have developed a new system using real-time PCR that sensitively detects the change in copy number of mtDNA compared with nDNA. In each sample, the DNA sequence coding 18S rRNA served as the nDNA reference in a run simultaneously with a mtDNA sequence. Small bowel collected 24 hours after 2 Gy or 4 Gy total body irradiation (TBI) exhibited increased levels of mtDNA compared with control mice. A 4 Gy dose produced a greater effect than 2 Gy. Similarly, in bone marrow collected 24 hours after 4 Gy or 7 Gy TBI, 7 Gy produced a greater response than 4 Gy. As a function of time, a greater effect was seen at 48 hours compared with 24 hours. In conclusion, we found that radiation increased the ratio of mtDNA:nDNA and that this effect seems to be tissue independent and seems to increase with radiation dose and duration following radiation exposure.

Response patterns of cytokines/chemokines in two murine strains after irradiation

PURPOSE To determine the plasma concentrations of acute responding cytokines/chemokines following 9-Gy ionizing radiation in C57BL/6 (radiation tolerant) and C3H/HeN (radiation sensitive) murine strains. METHODS AND MATERIALS Mice (5/group) received 9-Gy total body irradiation (TBI), and the plasma from each mouse was collected at 6h or 1, 2, 4, or 10 days after TBI. A multiplex bead array was used to assess the levels of 32 cytokines/chemokines in plasma to determine their common and strain-specific temporal responses. RESULTS The plasma levels of five cytokines/chemokines (Axl, FasL, ICAM-1, TARC, and TSLP) were beyond the detectable level. Five (VEGF, IL-2, IL-5, IL-17, and CD30) were unaffected by irradiation in either strain. Temporal patterns were similar in both murine strains for 10 of the cytokines tested, including G-CSF, IL-6, TCA-3, MCP-1, MIP-1γ, KC, CXCL 13, CXCL 16, MDC, and TIMP-1; the other 12 molecules (GM-CSF, IL-3, SCF, IL-1β, IL-4, IL-10, IL-12p70, MIP-1α, Eotaxin, TNF-α, sTNF-R1, and CD40) showed strain-specific response patterns. While a number of cytokines had temporal response patterns following TBI, the strains exhibited quantitatively different results. CONCLUSIONS The levels of 27 of the 32 plasma cytokines measured indicate the following: (1) different cytokine concentrations and temporal patterns in the two strains may partly explain different radiation sensitivities and sequelae following irradiation; (2) many of the cytokines/chemokines exhibit similar temporal responses in the two strains. These responses suggest the potential value of using a panel of cytokine/chemokine temporal patterns for radiation dosimetry. Although radiation doses will be difficult to quantitate due to the large variation in levels and temporal responses exhibited in the two murine strains, serial measurements of cytokines might help identify subjects exposed to radiation.

Fibroblast growth factor-peptide improves barrier function and proliferation in human keratinocytes after radiation.

PURPOSE Epidermal keratinocytes, which can be severely damaged after ionizing radiation (IR), are rapid turnover cells that function as a barrier, protecting the host from pathogenic invasion and fluid loss. We tested fibroblast growth factor-peptide (FGF-P), a small peptide derived from the receptor-binding domain of FGF-2, as a potential mitigator of radiation effects via proliferation and the barrier function of keratinocytes. METHODS AND MATERIALS Keratinocytes isolated from neonatal foreskin were grown on transwells. After being exposed to 0, 5, or 10 Gy IR, the cells were treated with a vehicle or FGF-P. The permeability of IR cells was assessed by using transepithelial electrical resistance (TEER) and a paracellular tracer flux of fluorescein isothiocyanate-conjugated bovine serum albumin (FITC-BSA) with Ussing chambers. The cell proliferation was measured with yellow tetrazolium salt (MTT) and tritiated thymidine ([3H]-TdR) assays. The phosphorylation of extracellular signal-regulated kinases (ERK) was measured in an enzyme-linked immunosorbent (ELISA)-like assay, and the proteins related to tight junctions (TJ) and adherens junctions (AJ) were examined with Western blotting. We used a mouse model to assess the ability of FGF-P to promote the healing of skin β burns created with a strontium applicator. RESULTS We found (1) FGF-P reduced the permeability of irradiated keratinocytes, as evidenced by increased TEER and decreased diffusion of FITC-BSA, both associated with the regulation of different proteins and levels of TJ and AJ; and (2) FGF-P enhanced the proliferation of irradiated keratinocytes, as evidenced by increased MTT activity and [3H]-TdR incorporation, which was associated with activation of the ERK pathway; and (3) FGF-P promoted the healing of skin β burns. CONCLUSIONS FGF-P enhances the barrier function, including up-regulation of TJ proteins, increases proliferation of human keratinocytes, and accelerates the healing of skin β burns. FGF-P is a promising mitigator that improves the proliferation and barrier function of keratinocytes after IR.

Radiation decreases murine small intestinal HCO3− secretion

Purpose: While secretagogue-induced diarrhea is rich in chloride (Cl−) and bicarbonate (HCO3 −) anions, little is known about diarrhea or its anionic composition following irradiation. We performed studies to characterize the differences between cyclic adenosine monophosphate (cAMP)-stimulated anion secretions in irradiated and non-irradiated mice. Materials and methods: HCO3 − secretion was examined in basal, cAMP-stimulated, and irradiated jejunal tissues from BALB/c (Bagg albino) mice. The abdomens of the mice were γ-irradiated using a caesium-137 source. Results: Ussing-chamber experiments performed in an HCO3−-containing, Cl−-free solution on the bath side showed inhibition of HCO3− in irradiated mice. Non-irradiated mice exhibited bumetanide-sensitive and insensitive current, while irradiated mice displayed bumetanide-sensitive current. pH-stat experiments showed inhibition of basal and cAMP-stimulated HCO3− secretions in irradiated mice. Immunohistochemistry and Western blot analysis displayed a sodium-bicarbonate cotransporter expression in the villus and not the crypt of non-irradiated mice, while its expression and protein levels decreased in irradiated mice. Conclusions: Anion secretions in irradiated mice, being primarily Cl− and minimally HCO3−, differ from that of secretagogue-induced anion secretions. Understanding anion loss will help us correct electrolyte imbalances, while reduced HCO3− secretion in the upper-gastrointestinal tract might also have implications for irradiation-induced nausea and vomiting.

Alteration of the Inflammatory Molecule Network After Irradiation of Soft Tissue

Inflammatory molecules (IMs) play an important role in ionizing radiation (IR)-induced soft tissue damage. The alteration of IMs as a function of time was studied with a protein array containing 62 IMs in mouse cutaneous soft tissues exposed to 30 Gy. The results showed that: (1) 2 days after irradiation, the levels of TGF-β1, MIP-1γ, IL-1α, and sTNF RI increased, while IGFBP-3, CXCL16, and IL-1β decreased in IR skin as compared to control skin; (2) 21 days after IR, TGF-β1, and MIP-1 γ, IL-1α remained high, while CXCL16 and IL-1β remained low; (3) 3 months after IR, the cytokine pattern exhibited reversals. The levels of MIP-1γ decreased, while VCAM-1, IGFBP-3, and TGF-β1 production increased. The data indicated that: (a) IMs change as a function of time after soft tissue irradiation; (b) changing IM levels may reflect the altered balance of the cytokine network, leading to imbalance or homeostasis; and (c) an antibody-based protein array can be used to assess multiple IMs simultaneously, making it useful for bulk screening for changes in tissue cytokine levels.

Radiation-Induced Elevation of Plasma DNA in Mice is Associated with Genomic Background

Genomic background helps determine sensitivity to TBI. Since the LD50/30 (the dose that causes death in half of exposed mice within 30 days) following TBI varies between murine strains, we tested four murine strains to determine whether TBI sensitivity was associated with different levels of circulating DNA after radiation. The LD50/30 for the mice we tested – BALB/c, NIH Swiss, C3H/HeN, and C57BL/6 – is approximately 5.8 ± 0.3, 7.3 ± 0.2, 7.4 ± 0.3, and 8.5 ± 0.3 Gy, respectively. We estimated the radiation dose at which circulating DNA reached a peak level (peakGy), and mice were subjected to different doses at 1.84 Gy/min. The peakGy was 6, 7, 9, and 9 Gy for BALB/c, NIH Swiss, C3H/HeN, and C57BL/6, which corresponds to each strain’s respective LD50/30. BALB/c, the most sensitive strain, had the lowest DNA concentration at peakGy, while C57BL/6, the most resistant strain, had the highest concentration at peakGy. At 7 Gy, the plasma DNA was approximately 3,754 ± 636, 8,238 ± 2,704, 9,773 ± 2,821, and 22,733 ± 5,914 ng/ml for BALB/c, NIH Swiss, C3H/HeN, and C57BL/6, respectively. These findings support our hypothesis that plasma DNA level is associated with genomic background.