Bridging the biology of the ovine TRALI model – human inflammatory cell responses to TRALI inducing supernatants

Background Transfusion-related acute lung injury (TRALI) is a serious and potentially fatal consequence of transfusion. A two-event TRALI model demonstrated date-of-expiry - day (D) 5 platelet (PLT) and D42 packed red blood cell (PRBC) supernatants (SN) induced TRALI in LPS-treated sheep. We have adapted a whole blood transfusion culture model as an investigative bridge between the ovine TRALI model human responses to transfusion. Methods A whole blood transfusion model was adapted to replicate the ovine model - specifically +/- 0.23μg/mL LPS as the first event and 10% SN volume (transfusion) as the second event. Four pooled SN from blood products, previously used in the TRALI ovine model, were investigated: D1-PLT, D5-PLT, D1-PRBC, and D42-PRBC. Fresh human whole blood (recipient) was mixed with combinations of LPS and BP-SN stimuli and incubated in vitro for 6 hrs. Addition of golgi plug enabled measurement of monocyte cytokine production (IL-6, IL-8, IL-10, IL-12, TNF-α, IL-1α, CXCL-5, IP-10, MIP-1α, MCP-1) using multi-colour flow cytometry. Responses for 6 recipients were assessed. Results In the presence of LPS, D42-PRBC-SN significantly increased monocyte IL-6 (P=0.031), IL-8 (P=0.016) and IL-1α (P=0.008) production compared to D1-PRBC-SN. This response to D42-PRBC-SN was LPS-dependent, and was not evident in non-LPSstimulated controls. This response was also specific to D42-PRBC-SN, as similar changes were not evident for the D5-PLT-SN, compared to the D1-PLT-SN, regardless of the presence of LPS. D5-PLT-SN significantly increased IL-12 production (P=0.024) compared to D1-PLT-SN. This response was again LPS-dependent. Conclusions These data demonstrate a novel two-event mechanism of monocyte inflammatory response that was dependent upon both the presence of date-of-expiry blood product SN and LPS. Further, these results demonstrate different cytokines responses induced by date-of-expiry PLT-SN and PRBC-SN. These data are consistent with the evidence from the ovine TRALI model, and enhancing its relevance to transfusion related changes in humans.

Phase III trial of gemcitabine and carboplatin versus mitomycin, ifosfamide, and cisplatin or mitomycin, vinblastine, and cisplatin in patients with advanced nonsmall cell lung carcinoma

BACKGROUND. The authors compared gemcitabine and carboplatin (GC) with mitomycin, ifosfamide, and cisplatin (MIC) or mitomycin, vinblastine, and cisplatin (MVP) in patients with advanced nonsmall cell lung carcinoma (NSCLC). The primary objective was survival. Secondary objectives were time to disease progression, response rates, evaluation of toxicity, disease-related symptoms, World Health Organization performance status (PS), and quality of life (QoL). METHODS. Three hundred seventy-two chemotherapy-naïve patients with International Staging System Stage III/IV NSCLC who were ineligible for curative radiotherapy or surgery were randomized to receive either 4 cycles of gemcitabine (1000 mg/m2 on Days 1, 8, and 15) plus carboplatin (area under the serum concentration-time curve, 5; given on Day 1) every 4 weeks (the GC arm) or MIC/MVP every 3 weeks (the MIC/MVP arm). RESULTS. There was no significant difference in median survival (248 days in the MIC/MVP arm vs. 236 days in the GC arm) or time to progression (225 days in the MIC/MVP arm vs. 218 days in the GC arm) between the 2 treatment arms. The 2-year survival rate was 11.8% in the MIC/MVP arm and 6.9% in the GC arm. The 1-year survival rate was 32.5% in the MIC/MVP arm and 33.2% in the GC arm. In the MIC/MVP arm, 33% of patients responded (4 complete responses [CRs] and 57 partial responses [PRs]) whereas in the GC arm, 30% of patients responded (3 CRs and 54 PRs). Nonhematologic toxicity was comparable for patients with Grade 3-4 symptoms, except there was more alopecia among patients in the MIC/MVP arm. GC appeared to produce more hematologic toxicity and necessitated more transfusions. There was no difference in performance status, disease-related symptoms, of QoL between patients in the two treatment arms. Fewer inpatient stays for complications were required with GC. CONCLUSIONS. The results of the current study failed to demonstrate any difference in efficacy between the newer regimen of GC and the older regimens of MIC and MVP. © 2003 American Cancer Society.

Randomized phase II study of cetuximab plus cisplatin/vinorelbine compared with cisplatin/vinorelbine alone as first-line therapy in EGFR-expressing advanced non-small-cell lung cancer

Background: The Lung Cancer Cetuximab Study is an open-label, randomized phase II pilot study of cisplatin and vinorelbine combined with the epidermal growth factor receptor (EGFR)-targeted monoclonal antibody cetuximab versus cisplatin and vinorelbine alone, in patients with advanced EGFR-expressing, non-small-cell lung cancer (NSCLC). End points of the study are activity, safety and pharmacokinetics. Patients and methods: Following randomization, for a maximum of eight cycles, patients received three-weekly cycles of cisplatin (80 mg/m2, day 1) and vinorelbine (25 mg/m2 on days 1 and 8) alone or following cetuximab treatment (initial dose 400 mg/m, followed by 250 mg/m2 weekly thereafter). Results: Eighty-six patients were randomly allocated to the study (43 per arm). Confirmed response rates were 28% in the cisplatin/vinorelbine arm (A) and 35% in the cetuximab plus cisplatin/vinorelbine arm (B). Median progression-free survival (PFS) was 4.6 months in arm A and 5.0 months in arm B, with PFS rates at 12 months of 0% and 15%, respectively. Median survival was 7.3 months in arm A and 8.3 months in arm B. The 24-month survival rates were 0% and 16%, respectively. The cetuximab combination was well tolerated. Conclusion: In the first-line treatment of advanced NSCLC, the combination of cetuximab plus cisplatin/vinorelbine demonstrated an acceptable safety profile and the potential to improve activity over cisplatin/vinorelbine alone. © 2007 European Society for Medical Oncology.

Gemcitabine and paclitaxel associated pneumonitis in non-small cell lung cancer : report of a phase I/II dose-escalating study

The aim of this phase I/II dose escalating study was to establish the maximum tolerated dose (MTD) of gemcitabine and paclitaxel given in combination in non-small cell lung cancer (NSCLC). 12 patients with stage IIIB and IV NSCLC received paclitaxel administered intravenously over 1 h followed by gemcitabine given over 30 min on days 1, 8 and 15 every 28 days. Pneumonitis was the principal side-effect observed with 4 patients affected. Of these, 1 experienced grade 3 toxicity after one cycle of treatment and the others had grade 2 toxicity. All 4 cases responded to prednisolone. No other significant toxicities were observed. Of the 8 evaluable patients, 3 had a partial response and 2 had minor responses. The study was discontinued due to this dose-limiting toxicity. The combination of paclitaxel and gemcitabine shows promising antitumour activity in NSCLC, however, this treatment schedule may predispose to pneumonitis. (C) 2000 Elsevier Science Ltd.

Neutrophil oxidative burst is primed by supernatant from stored red cells : implications for Transfusion-Related Acute Lung Injury (TRALI)

Aim/Background: Transfusion-related acute lung injury (TRALI) is a potentially fatal adverse transfusion reaction. It is hypothesised to occur via a two-insult mechanism: the recipient’s underlying co-morbidity in addition to the transfusion of blood products activate neutrophils in the lung resulting in damaged endothelium and capillary leakage. Neutrophil activation may occur by antibody or non-antibody related mechanisms, with the length of storage of cellular blood products implicated in the latter. This study investigated non-antibody mediated priming and/or activation of neutrophil oxidative burst. Methods: A cytochrome C reduction assay was used to assess priming and activation of neutrophil oxidative burst by pooled supernatant (SN) from day 1 (D1; n=75) and day 42 (D42; n=113) packed red blood cells (PRBC). Pooled PRBC-SN were assessed in parallel with PAF (priming), fMLP (activating), PAF + fMLP (priming + activating) and buffer only (negative) controls. Cytochrome C reduction was measured over 30min at 37oC (inclusive of 10min priming). Neutrophil activation by PRBC-SN was assessed cf. buffer only and neutrophil priming by PRBC-SN was assessed by co-incubation with fMLP cf. fMLP alone. One-way ANOVA; Newman-Keuls post-test; p<0.05; n=10 independent assays. Results: Neither D1- nor D42- PRBC-SN alone activated neutrophil oxidative burst. In addition, D1-PRBC-SN did not prime fMLP-activated neutrophil oxidative burst. D42-PRBC-SN did, however, prime neutrophils for subsequent activation of oxidative burst by fMLP, the magnitude of response being similar to PAF (a known neutrophil priming agonist). Conclusion: These findings are consistent with the two-insult mechanism of TRALI. Factors released into the SN during PRBC storage contributed to neutrophil priming synergistically with other neutrophil stimulating agonists. This implicates PRBC storage duration as a key factor contributing to non-immune neutrophil activation in the development of TRALI in patients with pre-disposing inflammatory conditions.

Effect of cell wall properties of plant tissue on porosity and shrinkage of dried apple

In this study cell wall properties; moisture distribution, stiffness, thickness and cell dimension have been taken into consideration. Cell wall stiffness dependent on complex combination of plant cell microstructures, composition and water holding capacity of the cell. In this work, some preliminary steps taken by investing cell wall properties of apple in order to predict change of porosity and shrinkage during drying. Two different types of apple cell wall characteristic were investigated to correlate with porosity and shrinkage after convective drying. A scanning electron microscope (SEM), 2N Intron, a pyncometer and image J software were used in order to measure and analyze cell characteristics, water dynamics, porosity and shrinkage. Cell stiffness of red delicious apple was found higher than granny smith apples. A significant relationship has found between cell wall characteristics and both heat and mass transfer. Consequently, evolution of porosity and shrinkage noticeably influenced during convective drying by the nature of cell wall. This study has brought better understanding of porosity and shrinkage of dried food stuff in microscopic (cell) level and would provide better insight to attain energy effective drying process and quality food stuff.

Interleukin-1beta induces human proximal tubule cell injury, alpha-smooth muscle actin expression and fibronectin production

BACKGROUND Tubulointerstitial lesions, characterized by tubular injury, interstitial fibrosis and the appearance of myofibroblasts, are the strongest predictors of the degree and progression of chronic renal failure. These lesions are typically preceded by macrophage infiltration of the tubulointerstitium, raising the possibility that these inflammatory cells promote progressive renal disease through fibrogenic actions on resident tubulointerstitial cells. The aim of the present study, therefore, was to investigate the potentially fibrogenic mechanisms of interleukin-1beta (IL-1beta), a macrophage-derived pro-inflammatory cytokine, on human proximal tubule cells (PTC). METHODS Confluent, quiescent, passage 2 PTC were established in primary culture from histologically normal segments of human renal cortex (N = 11) and then incubated in serum- and hormone-free media supplemented with either IL-1beta (0 to 4 ng/mL) or vehicle (control). RESULTS IL-1beta significantly enhanced fibronectin secretion by up to fourfold in a time- and concentration-dependent fashion. This was accompanied by significant (2.5- to 6-fold) increases in alpha-smooth muscle actin (alpha-SMA) expression, transforming growth factor beta (TGF-beta1) secretion, nitric oxide (NO) production, NO synthase 2 (NOS2) mRNA and lactate dehydrogenase (LDH) release. Cell proliferation was dose-dependently suppressed by IL-1beta. NG-methyl-l-arginine (L-NMMA; 1 mmol/L), a specific inhibitor of NOS, blocked NO production but did not alter basal or IL-1beta-stimulated fibronectin secretion. In contrast, a pan-specific TGF-beta neutralizing antibody significantly blocked the effects of IL-1beta on PTC fibronectin secretion (IL-1beta, 268.1 +/- 30.6 vs. IL-1beta+alphaTGF-beta 157.9 +/- 14.4%, of control values, P < 0.001) and DNA synthesis (IL-1beta 81.0 +/- 6.7% vs. IL-1beta+alphaTGF-beta 93.4 +/- 2.1%, of control values, P < 0.01). CONCLUSION IL-1beta acts on human PTC to suppress cell proliferation, enhance fibronectin production and promote alpha-smooth muscle actin expression. These actions appear to be mediated by a TGF-beta1 dependent mechanism and are independent of nitric oxide release.

T helper cell cytokine profiles following endurance exercise

Endurance exercise can cause immunosuppression and increase the risk of upper respiratory illness. The present study examined changes in the secretion of T helper (Th) cell cytokines after endurance exercise. Ten highly trained road cyclists [mean±SEM: age 24.2±1.7 years; height 1.82±0.02 m; body mass 73.8±2.0 kg; peak oxygen uptake 65.9±2.3 mL/(kg•min)] performed 2 h of cycling exercise at 90% of the second ventilatory threshold. Peripheral blood mononuclear cells were isolated and stimulated with phytohemagglutinin. Plasma cortisol concentrations and the concentration of Th1/Th2/Th17 cell cytokines were examined. Data were analyzed using both traditional statistics and magnitude-based inferences. Results revealed a significant decrease in plasma cortisol at 4–24 h postexercise compared with pre-exercise values. Qualitative analysis revealed postexercise changes in concentrations of plasma cortisol, IL-2, TNF, IL-4, IL-6, IL-10, and IL-17A compared with pre-exercise values. A Th1/Th2 shift was evident immediately postexercise. Furthermore, for multiple cytokines, including IL-2 and TNF (Th1), IL-6 and IL-10 (Th2), and IL-17 (Th17), no meaningful change in concentration occurred until more than 4 h postexercise, highlighting the duration of exercise-induced changes in immune function. These results demonstrate the importance of considering “clinically” significant versus statistically significant changes in immune cell function after exercise.

Gelatine methacrylamide-based hydrogels : an alternative three-dimensional cancer cell culture system

Modern cancer research requires physiological, three-dimensional (3-D) cell culture platforms, wherein the physical and chemical characteristics of the extracellular matrix (ECM) can be modified. In this study, gelatine methacrylamide (GelMA)-based hydrogels were characterized and established as in vitro and in vivo spheroid-based models for ovarian cancer, reflecting the advanced disease stage of patients, with accumulation of multicellular spheroids in the tumour fluid (ascites). Polymer concentration (2.5-7% w/v) strongly influenced hydrogel stiffness (0.5±0.2kPa to 9.0±1.8kPa) but had little effect on solute diffusion. The diffusion coefficient of 70kDa fluorescein isothiocyanate (FITC)-labelled dextran in 7% GelMA-based hydrogels was only 2.3 times slower compared to water. Hydrogels of medium concentration (5% w/v GelMA) and stiffness (3.4kPa) allowed spheroid formation and high proliferation and metabolic rates. The inhibition of matrix metalloproteinases and consequently ECM degradability reduced spheroid formation and proliferation rates. The incorporation of the ECM components laminin-411 and hyaluronic acid further stimulated spheroid growth within GelMA-based hydrogels. The feasibility of pre-cultured GelMA-based hydrogels as spheroid carriers within an ovarian cancer animal model was proven and led to tumour development and metastasis. These tumours were sensitive to treatment with the anti-cancer drug paclitaxel, but not the integrin antagonist ATN-161. While paclitaxel and its combination with ATN-161 resulted in a treatment response of 33-37.8%, ATN-161 alone had no effect on tumour growth and peritoneal spread. The semi-synthetic biomaterial GelMA combines relevant natural cues with tunable properties, providing an alternative, bioengineered 3-D cancer cell culture in in vitro and in vivo model systems.

An R2R3 MYB transcription factor determines red petal colour in an Actinidia (kiwifruit) hybrid population

Background Red colour in kiwifruit results from the presence of anthocyanin pigments. Their expression, however, is complex, and varies among genotypes, species, tissues and environments. An understanding of the biosynthesis, physiology and genetics of the anthocyanins involved, and the control of their expression in different tissues, is required. A complex, the MBW complex, consisting of R2R3-MYB and bHLH transcription factors together with a WD-repeat protein, activates anthocyanin 3-O-galactosyltransferase (F3GT1) to produce anthocyanins. We examined the expression and genetic control of anthocyanins in flowers of Actinidia hybrid families segregating for red and white petal colour. Results Four inter-related backcross families between Actinidia chinensis Planch. var. chinensis and Actinidia eriantha Benth. were identified that segregated 1:1 for red or white petal colour. Flower pigments consisted of five known anthocyanins (two delphinidin-based and three cyanidin-based) and three unknowns. Intensity and hue differed in red petals from pale pink to deep magenta, and while intensity of colour increased with total concentration of anthocyanin, no association was found between any particular anthocyanin data and hue. Real time qPCR demonstrated that an R2R3 MYB, MYB110a, was expressed at significant levels in red-petalled progeny, but not in individuals with white petals. A microsatellite marker was developed that identified alleles that segregated with red petal colour, but not with ovary, stamen filament, or fruit flesh colour in these families. The marker mapped to chromosome 10 in Actinidia. The white petal phenotype was complemented by syringing Agrobacterium tumefaciens carrying Actinidia 35S::MYB110a into the petal tissue. Red pigments developed in white petals both with, and without, co-transformation with Actinidia bHLH partners. MYB110a was shown to directly activate Actinidia F3GT1 in transient assays. Conclusions The transcription factor, MYB110a, regulates anthocyanin production in petals in this hybrid population, but not in other flower tissues or mature fruit. The identification of delphinidin-based anthocyanins in these flowers provides candidates for colour enhancement in novel fruits.

Red colouration in apple fruit is due to the activity of the MYB transcription factor, MdMYB10

Anthocyanin concentration is an important determinant of the colour of many fruits. In apple (Malus x domestica), centuries of breeding have produced numerous varieties in which levels of anthocyanin pigment vary widely and change in response to environmental and developmental stimuli. The apple fruit cortex is usually colourless, although germplasm does exist where the cortex is highly pigmented due to the accumulation of either anthocyanins or carotenoids. From studies in a diverse array of plant species, it is apparent that anthocyanin biosynthesis is controlled at the level of transcription. Here we report the transcript levels of the anthocyanin biosynthetic genes in a red-fleshed apple compared with a white-fleshed cultivar. We also describe an apple MYB transcription factor, MdMYB10, that is similar in sequence to known anthocyanin regulators in other species. We further show that this transcription factor can induce anthocyanin accumulation in both heterologous and homologous systems, generating pigmented patches in transient assays in tobacco leaves and highly pigmented apple plants following stable transformation with constitutively expressed MdMYB10. Efficient induction of anthocyanin biosynthesis in transient assays by MdMYB10 was dependent on the co-expression of two distinct bHLH proteins from apple, MdbHLH3 and MdbHLH33. The strong correlation between the expression of MdMYB10 and apple anthocyanin levels during fruit development suggests that this transcription factor is responsible for controlling anthocyanin biosynthesis in apple fruit; in the red-fleshed cultivar and in the skin of other varieties, there is an induction of MdMYB10 expression concurrent with colour formation during development. Characterization of MdMYB10 has implications for the development of new varieties through classical breeding or a biotechnological approach.

High temperature reduces apple fruit colour via modulation of the anthocyanin regulatory complex

The biosynthesis of anthocyanin in many plants is affected by environmental conditions. In apple (Malus×domestica Borkh.), concentrations of fruit anthocyanins are lower under hot climatic conditions. We examined the anthocyanin accumulation in the peel of maturing 'Mondial Gala' and 'Royal Gala' apples, grown in both temperate and hot climates, and using artificial heating of on-tree fruit. Heat caused a dramatic reduction of both peel anthocyanin concentration and transcripts of the genes of the anthocyanin biosynthetic pathway. Heating fruit rapidly reduced expression of the R2R3 MYB transcription factor (MYB10) responsible for coordinative regulation for red skin colour, as well as expression of other genes in the transcriptional activation complex. A single night of low temperatures is sufficient to elicit a large increase in transcription of MYB10 and consequently the biosynthetic pathway. Candidate genes that can repress anthocyanin biosynthesis did not appear to be responsible for reductions in anthocyanin content. We propose that temperature-induced regulation of anthocyanin biosynthesis is primarily caused by altered transcript levels of the activating anthocyanin regulatory complex.

Multiple repeats of a promoter segment causes transcription factor autoregulation in red apples

Mutations in the genes encoding for either the biosynthetic or transcriptional regulation of the anthocyanin pathway have been linked to color phenotypes. Generally, this is a loss of function resulting in a reduction or a change in the distribution of anthocyanin. Here, we describe a rearrangement in the upstream regulatory region of the gene encoding an apple (Malus x domestica) anthocyanin-regulating transcription factor, MYB10. We show that this modification is responsible for increasing the level of anthocyanin throughout the plant to produce a striking phenotype that includes red foliage and red fruit flesh. This rearrangement is a series of multiple repeats, forming a minisatellite-like structure that comprises five direct tandem repeats of a 23-bp sequence. This MYB10 rearrangement is present in all the red foliage apple varieties and species tested but in none of the white fleshed varieties. Transient assays demonstrated that the 23-bp sequence motif is a target of the MYB10 protein itself, and the number of repeat units correlates with an increase in transactivation by MYB10 protein. We show that the repeat motif is capable of binding MYB10 protein in electrophoretic mobility shift assays. Taken together, these results indicate that an allelic rearrangement in the promoter of MYB10 has generated an autoregulatory locus, and this autoregulation is sufficient to account for the increase in MYB10 transcript levels and subsequent ectopic accumulation of anthocyanins throughout the plant.

Sphingosine-1-phosphate, a novel second messenger involved in cell growth regulation and signal transduction, affects growth and invasiveness of human breast cancer cells

This review will focus on the role of sphingosine and its phosphorylated derivative sphingosine-1-phosphate (SPP) in cell growth regulation and signal transduction. We will show that many of the effects attributed to sphingosine in quiescent Swiss 3T3 fibroblasts are mediated via its conversion to SPP. We propose that SPP has appropriate properties to function as an intracellular second messenger based on the following: it elicits diverse cellular responses; it is rapidly produced from sphingosine by a specific kinase and rapidly degraded by a specific lyase; its concentration is low in quiescent cells but increases rapidly and transiently in response to the growth factors, fetal calf serum (FCS) and platelet derived growth factor (PDGF); it releases Ca2+ from internal sources in an InsP3-independent manner; and finally, it may link sphingolipid signaling pathways to cellular ras-mediated signaling pathways by elevating phosphatidic acid levels. The effects of this novel second messenger on growth, differentiation and invasion of human breast cancer cells will be discussed. © 1994 Kluwer Academic Publishers.

Chemotaxis and chemokinesis of human prostate tumor cell lines in response to human prostate stromal cell secretory proteins containing a nerve growth factor-like protein

The migration of three human prostate tumor epithelial cell lines (TSU-pr1, PC-3, DU-145) in response to secreted protein from a human prostate stromal cell line was investigated by using the modified blind-well Boyden chamber assay. Migrated cells were quantified by spectrophotometrically measuring the concentration of crystal violet stain extracted from their nuclei. Cell number was correlated linearly with the concentration of extracted crystal violet stain. All three tumor cell lines showed intrinsic migratory ability in the absence of chemoattractants, such that approximately 1-7% of plated cells migrated across the filter of the Boyden chambers during a 5-h incubation period. Prostate tumor cell migration was significantly enhanced (3-13-fold) in response to stromal cell secretory protein in a dose-dependent manner, whereas bovine serum albumin had no effect on stimulating tumor cell migration. Immunoprecipitation of the stromal cell secreted protein with a nerve growth factor antibody partially and significantly reduced its stimulatory activity for tumor cell migration. A Zigmond-Hirsch matrix assay of tumor cell migration in response to various concentration gradients of stromal cell secreted protein demonstrated both chemotaxis and chemokinesis by all three cell lines. These results are consistent with the stromal cell secretory protein stimulation of chemokinetic tumor cell migration through the capsule of the prostate. Outside of the prostate gland metastasis of tumor cells may occur by chemotaxis to preferential sites containing chemoattractants similar to or related to maintenance factors that can substitute for components of stromal cell secretory protein.