Sadasivan Vidyasagar

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.

Glucose stimulates calcium-activated chloride secretion in small intestinal cells

The sodium-coupled glucose transporter-1 (SGLT1)-based oral rehydration solution (ORS) used in the management of acute diarrhea does not substantially reduce stool output, despite the fact that glucose stimulates the absorption of sodium and water. To explain this phenomenon, we investigated the possibility that glucose might also stimulate anion secretion. Transepithelial electrical measurements and isotope flux measurements in Ussing chambers were used to study the effect of glucose on active chloride and fluid secretion in mouse small intestinal cells and human Caco-2 cells. Confocal fluorescence laser microscopy and immunohistochemistry measured intracellular changes in calcium, sodium-glucose linked transporter, and calcium-activated chloride channel (anoctamin 1) expression. In addition to enhancing active sodium absorption, glucose increased intracellular calcium and stimulated electrogenic chloride secretion. Calcium imaging studies showed increased intracellular calcium when intestinal cells were exposed to glucose. Niflumic acid, but not glibenclamide, inhibited glucose-stimulated chloride secretion in mouse small intestines and in Caco-2 cells. Glucose-stimulated chloride secretion was not seen in ileal tissues incubated with the intracellular calcium chelater BAPTA-AM and the sodium-potassium-2 chloride cotransporter 1 (NKCC1) blocker bumetanide. These observations establish that glucose not only stimulates active Na absorption, a well-established phenomenon, but also induces a Ca-activated chloride secretion. This may explain the failure of glucose-based ORS to markedly reduce stool output in acute diarrhea. These results have immediate potential to improve the treatment outcomes for acute and/or chronic diarrheal diseases by replacing glucose with compounds that do not stimulate chloride secretion.

Synthesis and anticancer potential of novel xanthone derivatives with 3,6-substituted chains.

In an effort to develop new drug candidates with enhanced anticancer activity, our team synthesized and assessed the cytotoxicity of a series of novel xanthone derivatives with two longer 3,6-disubstituted amine carbonyl methoxy side chains on either benzene ring in selected human cancer cell lines. An MTT assay revealed that a set of compounds with lower IC50 values than the positive control, 5-FU, exhibited greater anticancer effects. The most potent derivative (XD8) exhibited anticancer activity in MDA-MB-231, PC-3, A549, AsPC-1, and HCT116 cells lines with IC50 values of 8.06, 6.18, 4.59, 4.76, and 6.09μM, respectively. Cell cycle analysis and apoptosis activation suggested that the mechanism of action of these derivatives includes cell cycle regulation and apoptosis induction.

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.

An amino acid mixture mitigates radiation-induced gastrointestinal toxicity.

AbstractElectrolyte and nutrient absorption occur in villous epithelial cells. Radiation often results in reduced electrolyte and nutrient absorption, which leads to gastrointestinal toxicity. Therefore, the authors studied: (1) radiation-induced changes in glucose and amino acid absorption across ileal tissues and (2) the effect of amino acid mixtures on absorptive capacity. NIH Swiss mice were irradiated (0, 1, 3, 5, or 7 Gy) using a 137Cs source at 0.9 Gy min−1. Transepithelial short circuit current (Isc), dilution potential, and isotope flux determinations were made in Ussing chamber studies and correlated to plasma endotoxin and IL‐1&bgr; levels. Amino acids that increased electrolyte absorption and improved mucosal barrier functions were used to create a mitigating amino acid mixture (MAAM). The MAAM was given to mice via gastric gavage; thereafter, body weight and survival were recorded. A significant decrease in basal and glucose-stimulated sodium absorption occurred after 0, 1, 3, 5, and 7 Gy irradiation. Ussing chamber studies showed that paracellular permeability increased following irradiation and that the addition of glucose resulted in a further increase in permeability. Following irradiation, certain amino acids manifested decreased absorption, whereas others were associated with increased absorption. Lysine, aspartic acid, glycine, isoleucine, threonine, tyrosine, valine, tryptophan, and serine decreased plasma endotoxins were selected for the MAAM. Mice treated with the MAAM showed increased electrolyte absorption and decreased paracellular permeability, IL‐1&bgr; levels, and plasma endotoxin levels. Mice treated with MAAM also had increased weight gain and better survival following irradiation. The MAAM has immediate potential for use in mitigating radiation-induced acute gastrointestinal syndrome.

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.

An amino acid-based oral rehydration solution (AA-ORS) enhanced intestinal epithelial proliferation in mice exposed to radiation.

Destruction of clonogenic cells in the crypt following irradiation are thought to cause altered gastrointestinal function. Previously, we found that an amino acid-based oral rehydration solution (AA-ORS) improved gastrointestinal function in irradiated mice. However, the exact mechanisms were unknown. Electrophysiology, immunohistochemistry, qPCR, and Western blot analysis were used to determine that AA-ORS increased proliferation, maturation, and differentiation and improved electrolyte and nutrient absorption in irradiated mice. A single-hit, multi-target crypt survival curve showed a significant increase in crypt progenitors in irradiated mice treated with AA-ORS for six days (8.8 ± 0.4) compared to the saline-treated group (6.1 ± 0.3; P < 0.001) without a change in D0 (4.8 ± 0.1 Gy). The Dq values increased from 8.8 ± 0.4 Gy to 10.5 ± 0.5 Gy with AA-ORS treatment (P < 0.01), indicating an increased radiation tolerance of 1.7 Gy. We also found that AA-ORS treatment (1) increased Lgr5+, without altering Bmi1 positive cells; (2) increased levels of proliferation markers (Ki-67, p-Erk, p-Akt and PCNA); (3) decreased apoptosis markers, such as cleaved caspase-3 and Bcl-2; and (4) increased expression and protein levels of NHE3 and SGLT1 in the brush border membrane. This study shows that AA-ORS increased villus height and improved electrolyte and nutrient absorption.

Transition pattern and mechanism of B-lymphocyte precursors in regenerated mouse bone marrow after subtotal body irradiation.

Little is known about the effects of ionizing radiation on the transition and the related signal transduction of progenitor B cells in the bone marrow. Thus, using an NIH Swiss mouse model, we explored the impact of ionizing radiation on the early stage of B-cell development via an examination of the transition of CLP to pro-B to pre-B cells within bone marrow as a function of radiation doses and times. Our results showed that while the total number of bone marrow lymphoid cells at different stages were greatly reduced by subtotal body irradiation (sub-TBI), the surviving cells continued to transition from common lymphoid progenitors to pro-B and then to pre-B in a reproducible temporal pattern. The rearrangement of the immunoglobulin heavy chain increased significantly 1–2 weeks after irradiation, but no change occurred after 3–4 weeks. The rearrangement of the immunoglobulin light chain decreased significantly 1–2 weeks after sub-TBI but increased dramatically after 3–4 weeks. In addition, several key transcription factors and signaling pathways were involved in B-precursor transitions after sub-TBI. The data indicate that week 2 after irradiation is a critical time for the transition from pro-B cells to pre-B cells, reflecting that the functional processes for different B-cell stages are well preserved even after high-dose irradiation.

Ussing Chamber Technique to Measure Intestinal Epithelial Permeability.

Epithelial cells are polarized and have tight junctions that contribute to barrier function. Assessment of barrier function typically involves measurement of electrophysiological parameters or movement of nonionic particles across an epithelium. Here, we describe measurement of transepithelial electrical conductance or resistance, determination of dilution potential, and assessment of flux of nonionic particles such as dextran or mannitol, with particular emphasis on Ussing chamber techniques.

A New Flavonoid Regulates Angiogenesis and Reactive Oxygen Species Production

The tumor vascular system, which is critical to the survival and growth of solid tumors, has been an attractive target for anticancer research. Building on studies that show that some flavonoids have anticancer vascular effects, we developed and analyzed the flavonoid derivative R24 [3, 6-bis (2-oxiranylmethoxy)-9H-xanthen-9-one]. A CAM assay revealed that R24 disrupted neovascular formation; fewer dendrites were detected and overall dendritic length was shorter in the R24-treated chicken embryos. The antiproliferative effect of R24 was measured by MTT assay in A549 (lung cancer), AsPC-1 (pancreatic cancer), HCT-116 (colorectal cancer), and PC-3 (prostate cancer) cell lines. R24 reduced proliferation with an IC50 of 3.44, 3.59, 1.22, and 11.83 μM, respectively. Cell-cycle analysis and Annexin-V/propidium iodide staining showed that R24 induced apoptosis. In addition, R24 regulated intracellular ROS production in a dose-dependent manner. CM-H2DCFDA staining indicated that intracellular ROS production increased with the R24 dose. In summary, we found that R24 exhibits potent antiangiogenic and antiproliferative effects, induces apoptosis, and promotes ROS production.