Exosomes derived from stem cells from the apical papilla alleviate inflammation in rat pulpitis by upregulating regulatory T cells.

AIM: To evaluate the effects of exosomes derived from stem cells from the apical papilla (SCAP-Exos) in rats with experimentally induced pulpitis and the effects of SCAP-Exos on the conversion of regulatory T cells (Tregs) and methylation status of the Foxp3 locus in Tregs in vitro. METHODOLOGY: SCAP-Exos were isolated and identified using transmission electron microscopy, western blotting, and nanoparticle tracking analysis. Lipopolysaccharide was used to experimentally induced pulpitis in rats, and the effects of SCAP-Exos on the rats with pulpitis were detected using haematoxylin-eosin staining and immunofluorescence staining. CD4+ CD25- T cells were treated with different doses of SCAP-Exos, and flow cytometric analysis was used to assess the effects of SCAP-Exos on Treg proliferation and conversion. An enzyme-linked immunosorbent assay (ELISA) was used to evaluate the expression of interleukin 10 (IL-10). MethylTarget® technology was used to measure the methylation level of the Foxp3 locus in T cells. The expression levels of ten-eleven-translocation (Tet) 1, Tet2, and Tet3 in T cells were detected by real-time PCR and western blotting. RESULTS: SCAP-Exos had an elliptical vesicle-like structure with a diameter of approximately 143.7 nm and expressed the exosomal markers Alix and CD9. SCAP-Exo administration increased Treg accumulation in the inflamed dental pulp and alleviated inflammation in the dental pulp in vivo. SCAP-Exos promoted Treg conversion in vitro. Mechanistically, SCAP-Exos promoted Tet2-mediated Foxp3 demethylation to maintain the stable expression of Foxp3. CONCLUSIONS: SCAP-Exos promoted Treg conversion and effectively alleviated inflammation in the dental pulp of rats. This study shows that SCAP-Exos can regulate the local immune microenvironment to favour tissue regeneration, thus providing a potential novel strategy utilising SCAP-Exos as a cell-free approach to treat early inflammation of dental pulp in immature permanent teeth in the clinic.

A unified call to action from Australian nursing and midwifery leaders: ensuring that Black lives matter.

Nurses and midwives of Australia now is the time for change! As powerfully placed, Indigenous and non-Indigenous nursing and midwifery professionals, together we can ensure an effective and robust Indigenous curriculum in our nursing and midwifery schools of education. Today, Australia finds itself in a shifting tide of social change, where the voices for better and safer health care ring out loud. Voices for justice, equity and equality reverberate across our cities, our streets, homes, and institutions of learning. It is a call for new songlines of reform. The need to embed meaningful Indigenous health curricula is stronger now than it ever was for Australian nursing and midwifery. It is essential that nursing and midwifery leadership continue to build an authentic collaborative environment for Indigenous curriculum development. Bipartisan alliance is imperative for all academic staff to be confident in their teaching and learning experiences with Indigenous health syllabus. This paper is a call out. Now is the time for Indigenous and non-Indigenous nurses and midwives to make a stand together, for justice and equity in our teaching, learning, and practice. Together we will dismantle systems, policy, and practices in health that oppress. The Black Lives Matter movement provides us with a 'now window' of accepted dialogue to build a better, culturally safe Australian nursing and midwifery workforce, ensuring that Black Lives Matter in all aspects of health care.

Probability of intense precipitation from polarimetric GNSS radio occultation observations.

There is currently a gap in satellite observations of the moisture structure during heavy precipitation conditions, since infrared and microwave sounders cannot sense water-vapour structure near the surface in the presence of intense precipitation. Conversely, Global Navigation Satellite System (GNSS) radio occultations (RO) can profile the moisture structure with high precision and vertical resolution, but cannot indicate the presence of precipitation directly. Polarimetric RO (PRO) measurements have been proposed as a method to characterize heavy rain in GNSS RO, by measuring the polarimetric differential phase delay induced by large size hydrometeors. Previous studies have shown that the PRO polarimetric phase shift is sensitive to the path-integrated rain rate under intense precipitation scenarios, but there is no current method to invert PRO measurements into quantitative estimates of the path-averaged rain rate. In this manuscript, a probabilistic inversion approach to the GNSS PRO observables is proposed, where the GPM precipitation products are used for the construction of an a priori look-up table (LUT) database. The performance of the LUTs is assessed for use in the inversion of satellite-based GNSS PRO observations, based on synthetically generated PRO data of actual events, which correspond to co-locations between GNSS RO profiles and the TRMM observations. The synthetic data include end-to-end propagation effects of the polarimetric observables and a simple separation algorithm to isolate the hydrometeor component of the observation. The assessment results in agreement better than ±1 mm/hr between the reference LUT and the actual rain statistics of the synthetic data, proving the suitability of the GPM-based probabilistic inversion tool. These findings indicate that the GNSS PRO products are capable of extending the current GNSS RO ones by associating indications of rain-rate probabilities at different altitudes, at ∼250 m vertical resolution and under intense precipitation scenarios with the standard vertical thermodynamic profiles.

Effects of continuous low dose infusion of lipopolysaccharide on inflammatory responses, milk production and milk quality in dairy cows.

The objective of this study was to evaluate the effects of continuous low dose infusion of lipopolysaccharide (LPS) on inflammatory responses and milk production and quality in lactating dairy cows. Eight Holstein cows were assigned to two treatments in a cross-over experimental design. Cows were infused intravenously either with saline solution or with saline solution containing LPS from Escherichia coli O111:B4 at a dose of 0.01 µg LPS/kg body weight for approximately 6 hr each day during a seven-day trial. The clinical symptoms and milk production performance were observed. Milk samples were analysed for conventional components, fatty acids and amino acids. And jugular vein and mammary vein plasma samples were analysed for concentrations of cytokines and acute phase proteins. LPS infusion decreased feed intake and milk yield. An increase in body temperature was observed after LPS infusion. LPS infusion also increased plasma concentrations of interleukin-1ß, serum amyloid A, LPS-binding protein, C-reactive protein and haptoglobin. LPS infusion decreased the contents of some fatty acids, such as C17:1, C18:0, C18:1n9 (trans) and C18:2n6 (trans), and most amino acids except for methionine, threonine, histidine, cysteine, tyrosine and proline in the milk. The results indicated that a continued low dose infusion of LPS can induce an inflammatory response, decrease milk production and reduce milk quality.

Combined effects of oleic, linoleic and linolenic acids on lactation performance and the milk fatty acid profile in lactating dairy cows.

The potential combined effects of oleic, linoleic and linolenic acids supplementation on lactation performance and the milk fatty acid (FA) profile in dairy cows have not been well investigated. Our objective was to examine the effects of supplementation with a combination of these FA as well as the effects of removing each from the combination on lactation performance and the milk FA profile in dairy cows. Eight Holstein cows (101±11 days in milk) received four intravenously infused treatments in a 4×4 Latin square design, and each period lasted for 12 days which consisted of 5 days of infusion and 7 days of recovery. The control treatment (CTL) contained 58.30, 58.17 and 39.96 g/day of C18 : 1 cis-9; C18 : 2 cis-9, cis-12; and C18 : 3 cis-9, cis-12, cis-15, respectively. The other three treatments were designated --C18 : 1 (20.68, 61.17 and 41.72 g/day of C18 : 1 cis-9; C18 : 2 cis-9, cis-12; and C18 : 3 cis-9, cis-12, cis-15, respectively), -C18 : 2 (61.49, 19.55 and 42.13 g/day of C18 : 1 cis-9; C18 : 2 cis-9, cis-12; and C18 : 3 cis-9, cis-12, cis-15, respectively) and -C18 : 3 (60.89, 60.16 and 1.53 g/day of C18 : 1 cis-9; C18 : 2 cis-9, cis-12; and C18 : 3 cis-9, cis-12, cis-15, respectively). Dry matter intake and lactose content were not affected by the treatments, but the milk protein content was lower in cows treated with -C18 : 2 than that in CTL-treated cows. Milk yield as well as milk fat, protein and lactose yields were higher in cows treated with -C18 : 3 than the yields in CTL-treated cows, and these yields increased linearly as the unsaturation degree of the supplemental FA decreased. Compared with the CTL treatment, the -C18 : 2 treatment decreased milk C18 : 2 cis-9 content (by 2.80%) and yield (by 22.12 g/day), and the -C18 : 3 treatment decreased milk C18 : 3 cis-9, cis-12, cis-15 content (by 2.72%) and yield (by 22.33 g/day). In contrast, removing C18 : 1 cis-9 did not affect the milk content or yield of C18 : 1 cis-9. The -C18 : 2-treated cows had a higher C18 : 1 cis-9 content and tended to have a higher C18 : 1 cis-9 yield than CTL-treated cows. The yields of C8 : 0, C14 : 0 and C16 : 0 as well as

Autophagy mediated by arginine depletion activation of the nutrient sensor GCN2 contributes to interferon-γ-induced malignant transformation of primary bovine mammary epithelial cells.

Autophagy has been linked to the regulation of both the prevention and progression of cancer. IFN-γ has been shown to induce autophagy in multiple cell lines in vitro. However, whether IFN-γ can induce autophagy and whether autophagy promotes malignant transformation in healthy lactating bovine mammary epithelial cells (BMECs) remain unclear. Here, we provide the first evidence of the correlation between IFN-γ treatment, autophagy and malignant transformation and of the mechanism underlying IFN-γ-induced autophagy and subsequent malignant transformation in primary BMECs. IFN-γ levels were significantly increased in cattle that received normal long-term dietary corn straw (CS) roughage supplementation. In addition, an increase in autophagy was clearly observed in the BMECs from the mammary tissue of cows expressing high levels of IFN-γ. In vitro, autophagy was clearly induced in primary BMECs by IFN-γ within 24 h. This induced autophagy could subsequently promote dramatic primary BMEC transformation. Furthermore, we found that IFN-γ promoted arginine depletion, activated the general control nonderepressible-2 kinase (GCN2) signalling pathway and resulted in an increase in autophagic flux and the amount of autophagy in BMECs. Overall, our findings are the first to demonstrate that arginine depletion and kinase GCN2 expression mediate IFN-γ-induced autophagy that may promote malignant progression and that immunometabolism, autophagy and cancer are strongly correlated. These results suggest new directions and paths for preventing and treating breast cancer in relation to diet.

Effects of different model diets on milk composition and expression of genes related to fatty acid synthesis in the mammary gland of lactating dairy goats.

This study examined the effects of different roughage diets on milk composition and the expression of key genes associated with fatty acid (FA) synthesis in the mammary gland of lactating dairy goats. Eight multiparous lactating goats (body weight=43.6±2.5kg, 90±12 d in milk) fitted with external pudic artery and subcutaneous abdominal vein catheters were assigned to 2 treatments in a crossover design. The goats were fed different roughage diets with a similar concentrate-to-roughage ratio. The diets were (1) a high-quality roughage treatment (HQR) containing 28.5% Chinese wildrye hay, 19% corn silage, 9.5% alfalfa, and 43% concentrate or (2) a low-quality roughage treatment (LQR) containing 28% Chinese wildrye hay, 28% corn stover, and 44% concentrate, on a dry matter basis. Each feeding period lasted 21 d. The first 18 d served as an adaptation period, and the last 3 d served as a sample collection period. Dry matter intake, milk yield, and milk composition were measured. Milk and blood samples were collected for FA analysis. Mammary gland biopsies were performed after milking on the last day of each period and the tissues were analyzed for the mRNA expression of acetyl-coenzyme A carboxylase-α (ACACA), FA synthase (FASN), stearoyl CoA desaturase (SCD), and lipoprotein lipase (LPL). Dry matter intake and milk yield were not affected by the treatments. Milk fat (3.16 vs. 2.96%) and protein (2.99 vs. 2.89%) contents were higher in HQR goats than in LQR goats, and milk fat yield tended to be higher in HQR goats (16.7 vs. 15.1g/d). Milk FA composition was not different between treatments, except for C18:3n-3 (0.27 vs. 0.15g/100g). Compared with LQR goats, HQR goats had a higher vein concentration of total FA (0.62 vs. 0.44mg/mL). In HQR goats, the mammary balance of total FA increased (9.17 vs. 5.51g/d), whereas the clearance rate of total FA decreased (103.03 vs. 138.25 L/d). No differences were found in mammary blood flow, artery concentration, and mammary uptake of FA between treatments. Compared with LQR, the expression of FASN and ACACA tended to be increased by 20 and 18%, and the expression of LPL and SCD were increased by 39 and 50% in HQR, respectively. The results demonstrated that diets with HQR can increase milk fat content and yield as well as the expression of LPL and SCD in the mammary gland of dairy goats.

Antimicrobial activity of the essential oil of Cymbopogon citratus (DC) Stapf. on Staphylococcus spp., Streptococcus mutans and Candida spp. / Atividade antimicrobiana do óleo essencial de Cymbopogon citratus (DC) Stapf. sobre Staphylococcus spp., Streptococcus mutans e Candida spp.

Medicinal plants with fungicide action, antibacterial and anti-inflammatory effects are under investigation. The main purpose of this work was to evaluate the antimicrobial activity of the essential oil from Cymbopogon citratus (DC) Stapf. on strains of Staphylococcus spp., Streptococcus mutans and Candida spp. with planktonic and biofilm growth. To study the micro-organisms in planktonic cells, the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) were determined by using 9 clinical strains for each species and 1 ATCC (American Type Culture Collection) from C. albicans, C. tropicalis, C. glabrata, S. aureus, S. epidermidis and S. mutans. In order to evaluate the effects of the essential oils on biofilms, strains of S. aureus (ATCC 6538), S. mutans (ATCC 35688) and C. albicans (ATCC 18804) were used. The biofilm was formed on acrylic resin discs with isolated micro-organisms or in associations. The number of colony-forming-units (CFU) obtained in each biofilm (CFU/ml) was submitted to Student's t statistical test. The results demonstrated that the essential oil of Cymbopogon citratus showed microbiostatic and microbicidal activity against all tested strains. The average CFU/ml for the biofilm of S. aureus, S. mutans and C. albicans, whether isolated or in association, was lower in the group treated with essential oil than in the control group.

As ações fungicida, antibacteriana e anti-inflamatória são propriedades importantes que vêm sendo investigadas em espécies medicinais. O objetivo do presente trabalho foi o de avaliar a atividade antimicrobiana do óleo essencial de Cymbopogon citratus (DC) Stapf. (capim-limão) sobre cepas de Staphylococcus spp., Streptococcus mutans e Candida spp. em crescimento planctônico e em biofilme. Para estudo dos micro-organismos em crescimento planctônico foram determinadas a Concentração Inibitória Mínima (CIM) e a Concentração Microbicida Mínima (CBM) de 9 cepas clínicas e, para cada espécie, uma ATCC (American Type Culture Collection) de: C. albicans, C. tropicalis, C. glabrata, S. aureus, S. epidermidis e S. mutans. Para a avaliação dos efeitos dos óleos essenciais em biofilme foram utilizadas cepas padrão de S. aureus (ATCC 6538), S. mutans (ATCC 35688) e C. albicans (ATCC 18804). O biofilme foi formado em corpos-de-prova de resina acrílica com os micro-organismos isolados ou em associações. O número de unidades formadoras de colônias obtidas em cada biofilme (UFC/ mL) foi submetido ao teste t de Student. Os resultados demonstraram que o óleo essencial de Cymbopogon citratus apresentou atividade microbiostática e microbicida para todas as cepas analisadas. As médias de UFC/ml para o biofilme de S. aureus, S. mutans e C. albicans, isolados ou associados, foram menores no grupo tratado com óleo essencial em relação ao grupo controle.

RAPD-based genetic diversities and correlation with morphological traits in Camellia (Theaceae) cultivars in China.

Camellia is an economically important ornamental plant that has many uses, such as in beverages, foods and medicines. We examined 15 Camellia cultivars in Wenzhou, China, using RAPD markers and measurements of three traits (petal color, flower diameter, blooming period). PCR amplification with 15 random primers produced 1935 bands, observed at 88 amplification loci; 77% of the amplified loci were polymorphic, with a mean of 4.5 polymorphic loci per primer. The similarity coefficient ranged from 0.5419 to 0.7933 among the 15 samples; the lowest value was between Manao (C. reticulata) and Feibai FR (C. japonica), and the largest value was between Chidan (C. japonica) and Yuanyang FG (C. japonica). Cluster analysis divided the 15 cultivars into two groups at the similarity coefficient of 0.65. A correlation was found between RAPD markers and petal color in the first group. No correlation was found between RAPD markers and the other traits (flower diameter, blooming period). This study provides information useful for the identification, classification, phylogenesis, and breeding of Camellia cultivars.

Multivariate analysis for stormwater quality characteristics identification from different urban surface types in macau.

Statistical analysis of stormwater runoff data enables general identification of runoff characteristics. Six catchments with different urban surface type including roofs, roadway, park, and residential/commercial in Macau were selected for sampling and study during the period from June 2005 to September 2006. Based on univariate statistical analysis of data sampled, major pollutants discharged from different urban surface type were identified. As for iron roof runoff, Zn is the most significant pollutant. The major pollutants from urban roadway runoff are TSS and COD. Stormwater runoff from commercial/residential and Park catchments show high level of COD, TN, and TP concentration. Principal component analysis was further done for identification of linkages between stormwater quality and urban surface types. Two potential pollution sources were identified for study catchments with different urban surface types. The first one is referred as nutrients losses, soil losses and organic pollutants discharges, the second is related to heavy metals losses. PCA was proved to be a viable tool to explain the type of pollution sources and its mechanism for different urban surface type catchments.

Effect of an angiotensin II receptor blocker and two angiotensin converting enzyme inhibitors on transforming growth factor-beta (TGF-beta) and alpha-actomyosin (alpha SMA), important mediators of radiation-induced pneumopathy and lung fibrosis.

Progressive, irreversible fibrosis is one of the most clinically significant consequences of ionizing radiation on normal tissue. When applied to lungs, it leads to a complication described as idiopathic pneumonia syndrome (IPS) and eventually to organ fibrosis. For its high mortality, the condition precludes treatment with high doses of radiation. There is widespread interest to understand the pathogenetic mechanisms of IPS and to find drugs effective in the prevention of its development. This report summarizes our experience with the protective effects of L 158,809, an angiotensin II (ANG II) receptor blocker, and two angiotensin converting enzyme (ACE) inhibitors in the development of IPS and the role of transforming growth factor beta (TGF-beta) and of alpha-actomyosin (alpha SMA) in pathogenesis of radiation induced pulmonary fibrosis in an experimental model of bone marrow transplant (BMT). Male WAG/Riji/MCV rats received total body irradiation and a regimen of cyclophosphamide (CTX) in preparation for bone marrow transplant. While one group of animals remained untreated, the remainders were subdivided into three groups, each of them receiving either the ANG II receptor blocker or one of the two ACE inhibitors (Captopril or Enalapril). Each of the three drugs was administered orally from 11 days before the transplant up to 56 days post transplant. At sacrifice time the irradiated rats receiving only CTX showed a chronic pneumonitis with septal fibrosis and vasculitis affecting, in particular, small caliber pulmonary arteries and arterioles. Their lung content of hydroxyproline was also markedly elevated in association with the lung concentrations of thromboxane (TXA2) and prostaglandin (PGI(2)), (two markers of pulmonary endothelial damage). A significant increase of alpha actomyosin staining was observed in vessels, septa and macrophages of the same animals which also overexpressed TGF-beta. When L 158,809, Captopril and Enalapril were added to the radiation and cytoxan treatment, a significant amelioration of the histological damage as well as the overexpression of alpha SMA was observed. Lung concentrations of hydroxyproline, PGI(2), TXA2 and TGF-beta were also observed in these animals so that the values of these compounds were closer to those measured in untreated control rats than to their irradiated and cytoxan treated counterparts. Angiotensin II plays an important role in the regulation of TGF-beta and alpha SMA, two proteins involved in the pathogenesis of pulmonary fibrosis. The finding that ACE inhibitors or ANG II receptor blockers protect the lungs from radiation induced pneumonitis and fibrosis reaffirms the role that ANG II plays in this inflammatory process and suggests an additional indication of treatment of this condition, thus opening a new potential pharmacologic use of these drugs.

Enhanced photocatalytic degradation of VOCs using Ln3+-TiO2 catalysts for indoor air purification.

Two types of lanthanide ion-doped titanium dioxide (Ln3+-TiO2) catalysts including La3+-TiO2 and Nd3+-TiO2 were prepared by a sol-gel method. The effects of the lanthanide ion doping on the crystal structure, surface area, adsorption properties, pore size distribution, and surface chemical state of the catalysts were investigated by means of XRD, BET, and XPS. As results, the crystal size decreased significantly, while the specific surface area, t-plot total surface area, micropore volume, and the total pore volume increased owing to the lanthanide ion doping. The nitrogen adsorption-desorption isotherms of the catalysts showed that the N2 adsorption ability of the Ln3+-TiO2 catalysts was better than the TiO2 catalyst. Among them, the 0.7% Ln3+-TiO2 catalysts demonstrated the highest adsorption ability. The photocatalytic activity of the catalysts was investigated in the experiments of the photocatalytic degradation of benzene, toluene, ethylbenzene and o-xylene (BTEX) in a gaseous phase. The photocatalytic efficiency of the TiO2 catalysts with the lanthanide ion doping was remarkably enhanced by BTEX removal. The 1.2% Ln3+-TiO2 catalysts achieved the highest photocatalytic activity. The enhanced photodegradation of BTEX is possibly due to the improved adsorption ability and the enhanced electron-hole pairs separation due to the presence of Ti3+ on the surface of Ln3+-TiO2 catalysts and the electron transfer between the conduction band/defect level and lanthanide crystal field state.

The "primitive" microaerophile Giardia intestinalis (syn. lamblia, duodenalis) has specialized membranes with electron transport and membrane-potential-generating functions.

Here it is shown that the flagellated protozoon Giardia intestinalis, commonly regarded as an early branching eukaryote because of its lack of mitochondria, has membraneous structures that partition the cationic, membrane-potential-sensitive fluorophore rhodamine 123. This organism also reduces a tetrazolium fluorogen at discrete plasma-membrane-associated sites. That these functions occur in distinctive specialized membrane systems supports the growing evidence that G. intestinalis may not be primitive, but is derived from an aerobic, mitochondria-containing flagellate.

Effects of unfractionated heparin, low-molecular-weight heparin, and heparinoid on thromboelastographic assay of blood coagulation.

Thromboelastography (TEG) has been used increasingly as an intraoperative hemostasis monitoring device. Low-molecular-weight heparins are given increasingly to reduce the development of antibodies against the heparin-platelet factor 4 complex, and heparinoids are given to patients who have developed the antibody. We studied the effect of unfractionated heparin, a low-molecular-weight heparin (enoxaparin sodium [Lovenox]), and a heparinoid (danaparoid sodium [Orgaran]) on blood clotting assayed with TEG (TEG clotting) in vitro and the efficacy of protamine sulfate and heparinase for reversing the effect. Heparin, enoxaparin, and danaparoid all caused a dose-dependent inhibition of TEG clotting of normal blood. Concentrations of enoxaparin and danaparoid that totally inhibited TEG clotting only minimally prolonged the activated partial thromboplastin time. While inhibition of TEG clotting by heparin and enoxaparin was reversed by protamine sulfate and heparinase, inhibition by danaparoid was reversed only by heparinase. Abnormal TEG clotting was observed in patients receiving enoxaparin whose plasma level of the drug was more than 0.1 antiXa U/mL. However, the degree of TEG abnormality did not always coincide with plasma levels of the drug.

Mechanism of captopril toxicity to a human mammary ductal carcinoma cell line in the presence of copper.

Captopril (D-3-mercapto-2-methylpropanoyl-L-proline) is an angiotensin converting enzyme (ACE) inhibitor, used widely in the treatment of hypertension and congestive heart failure. Captopril also inhibits proliferation of a variety of cell types, including several lacking ACE and renin acitvity. We have previously demonstrated that human mammary ductal carcinoma cells are among the cell types whose mitotic activity is inhibited by captopril. In those cells, captopril also reduces estrogen receptor (ER) and increases progesterone receptor (PR) concentrations. The present study evaluated the mechanism of captopril's antiproliferative action in an ER/PR-negative human mammary ductal carcinoma cell line, Hs578T. Cells grown in a 10% serum medium showed negligible changes in the presence of captopril alone. However, in the presence of subphysiologic concentrations of copper salts or copper-loaded ceruloplasmin, captopril caused a dose-dependent reduction in cell number, thymidine incorporation and mitochondrial dehydrogenase activity. In contrast, iron salts and iron-saturated transferrin had no effect on captopril activity. Catalase and horseradish peroxidase nullified the cytotoxic effects of captopril/Cu++, whereas H2O2 mimicked those effects. These data are consistent with the notion of a copper-catalyzed oxidation of captopril, leading to the generation of H2O2 as the cytotoxin to this clinically important cell type.

Performance characteristics of a new synthetic APTT reagent.

Performance characteristics of a totally synthetic activated partial thromboplastin time (APTT) reagent, recently available commercially, were evaluated and compared with a rabbit-brain extracted reagent. We found that the synthetic reagent, Synthasil, returned significantly higher normal APTT values than the brain-extracted reagent, Thrombosil. APTT ratios (APTT patients/normal mean APTT), yielded by Synthasil were higher in the majority of patients receiving heparin therapy. Synthasil also returned longer APTT values than did Thrombosil on normal plasma spiked with heparin. On patients with lupus anticoagulants, APTTs assayed with Synthasil were generally longer than with Thrombosil. However, the differences disappeared when APTT values were converted to ratios. Factors XII-, XI-, IX- and VIII-deficient plasmas supplemented with normal plasma to yield activities of 2-50%, generally gave longer APTTs with Synthasil than with Thrombosil. However, this was not always the case on plasmas from haemophilias A and B patients. No reduction in Synthasil activity was noted after the reagent had been left at 24 degrees C for 28 days.

Stability of plasma for add-on PT and APTT tests.

We conducted studies to determine at what time point an add-on prothrombin time (PT) or activated partial thromboplastin time (APTT) test can be honored on specimens that have been received in the laboratory hours earlier without yielding results with clinically significant differences from those if the test had been performed on the original unstored plasma. PT and APTT tests were performed on blood samples from 20 healthy subjects, 30 patients receiving warfarin, and 30 patients receiving heparin anticoagulation therapy. The tests were performed on plasma prepared initially after the samples were obtained. The same tests were assayed on plasma that had been left on spun-down blood cells at room temperature for 2, 4, and 8 hours. We found that the PT of the majority of plasma samples from healthy subjects and from patients receiving oral anticoagulant therapy tended to become shorter on storage. However, the difference in PT values was small and had no clinical significance. In most cases, the APTT values for the stored plasma from healthy subjects tended to increase with time. Except in one specimen in which the 8-hour add-on APTT was 1.2 seconds longer than the APTT result for the original sample, all others had APTT results less than 1.2 seconds longer than the original values. In patients receiving heparin, the differences in APTT values between the initial and add-on tests were larger than those observed for healthy subjects. However, those differences are not beyond what we would accept for duplicate checks for heparinized samples with high APTT values. Unlike samples from healthy subjects, there was no obvious trend of time-related prolongation of the APTT in heparinized plasma. These results led us to believe that within an 8-hour period and with plasma on spun-down cells at room temperature, add-on tests for PT and APTT could be performed with results similar to what would be obtained from testing unstored samples.

Annexin I in fibrotic rat lung and cultured lung fibroblasts following irradiation.

Radiation-induced lung fibrosis is a result of collagen accumulation in the interstitium, partly due to increased collagen synthesis by fibroblasts. One feature of active collagen synthesis is increased membrane trafficking in the fibroblasts. A group of proteins called annexins is believed to play a regulatory role in membrane fusion and exocytosis. Therefore, increased annexin activity might be expected in the fibrotic lung. We tested this hypothesis by measuring annexin I levels, hydroxyproline content and ultrastructural changes in radiation-induced pulmonary fibrosis in rat. Three months after a single exposure to 30 Gy of X-rays to the right hemithorax, the right lung of the rat was atrophied and fibrotic with a concomitant increase in size of the shielded left lung. Electron micrographs revealed that the irradiated lung was ladened with interstitial collagen fibrils, with increased number of fibroblasts amongst them. Hydroxyproline concentration in the irradiated lung was nearly twice that in the sham-irradiated lung. Annexin I in the irradiated lung, on the other hand, was markedly reduced, and barely detectable on immunoblots. Since increased annexin I might precede enhanced collagen production, we also measured annexin I levels in rat lungs 3 days after 30 Gy irradiation and correlated that with hydroxyproline concentration. We found no appreciable difference in annexin I levels and hydroxyproline content between sham-irradiated and irradiated lungs at 3 days. To determine whether annexin I levels in cultured fibroblasts were altered by irradiation, we assayed annexin I in cultured rat lung fibroblasts 3 days after 0.10 Gy exposure, with concomitant measurement of 14C-proline incorporation. The annexin I level in fibroblasts irradiated with 10 Gy X-rays was 55% higher than in sham-irradiated fibroblasts. However, incorporation of 14C-proline into collagenase-sensitive macromolecules in the culture medium and extracellular matrix was not different between these two groups of cells. These data demonstrate a radiation-induced increase in immunoreactive annexin I in cultured lung fibroblasts, but fail to support the hypothesis of a positive correlation between annexin I concentration and fibrosis in irradiated rat lung.

Modified APC-resistance test: variable ratios with respect to source of factor V-deficient plasma.

A single point mutation of the factor V (FV) gene, leading to the substitution Arg506Gln in the FV molecule (FV-Leiden) and hence resistance to its breakdown by activated protein C (APC), is the most prevalent risk factor for venous thrombosis in the Caucasians. A ratio determined by activated partial thromboplastin time (APTT) of test plasma in the presence or absence of exogenous APC (the APC ratio), is the method widely used to screen individuals with this risk factor for thrombosis. Because of functional defects of vitamin K-dependent clotting factors in patients on oral anticoagulant therapy, this method cannot be applied to those patients without modification. One modification is to mix test plasma (1:5 or 1:10) with FV-deficient plasma so that 80-90% of functioning vitamin K-dependent factors are supplied by the FV-deficient plasma. Even with 10-20% of FV in the mixture, APC-resistance still can be demonstrated. In this report, we present our results of the modified APC-sensitivity assay using FV-deficient plasma from different commercial sources. APC ratios determined by the original method in which test plasma is not mixed with FV-deficient plasma can be significantly different from those determined by the modified method in which test plasma is diluted 1:5 with FV-deficient plasma. This difference between methods was observed not only in normal individuals, but also in FV-Leiden positive individuals, and in patients on warfarin therapy. Further, APC ratios varied significantly depending on the commercial source of the FV-deficient plasma. The modified method is apparently suitable to identify APC-resistance in patients on warfarin therapy, as well as in individuals not receiving anticoagulant treatment. However, one must be aware that APC-resistance ratios obtained with the modified method are likely to be different from those established with the original method, and the source of FV-deficient plasma can be a factor influencing the ratios in the former cases.