Analysis of potential contamination factors in musculoskeletal tissues

Tissue Banks have become the main source for bone grafts, due to preference for homologous tissues. Notwithstanding the use of aseptic techniques for procurement of tissues and judicious selection of donors, microorganisms are frequently found in procured bones. Purpose of this study is to evaluate the factors that increase safety of procurement and minimize discard of procured tissues. Microbiological contamination was analyzed in 1271 musculoskeletal tissues removed from 138 multi-organ donors over a period extending from 2006 to 2016. Effects of various risk factors related with contamination were estimated using a logistic regression model. Microbiological contamination rate in the tissues was 17.1%; low pathogenic microorganisms were cultivated in 12.9% of the tissues, while highly pathogenic ones were cultivated in 4.2% of the tissues. Evolution of one single team was monitored during that period, verifying a fall in the general contamination level from 22.5 to 9.2%. Absence of antibiotics increased low pathogenic contamination risk. Every additional day in intensive care unit (ICU) increased the risk of highly pathogenic contamination. Time elapsed between death and the beginning of removal procedures was found to be relevant for both low pathogenic and highly pathogenic microorganisms. Among the studied factors, the following contributed for a significant increase in contamination by microorganisms in removed tissues: lack of use of prophylactic antibiotic therapy in donors, quantity of removed tissues, length of admission in ICU and the time elapsed between aortic clamping and beginning of the removal procedure.     

Stretching of the back improves gait, mechanical sensitivity and connective tissue inflammation in a rodent model

The role played by nonspecialized connective tissues in chronic non-specific low back pain is not well understood. In a recent ultrasound study, human subjects with chronic low back pain had altered connective tissue structure compared to human subjects without low back pain, suggesting the presence of inflammation and/or fibrosis in the low back pain subjects. Mechanical input in the form of static tissue stretch has been shown in vitro and in vivo to have anti-inflammatory and anti-fibrotic effects. To better understand the pathophysiology of lumbar nonspecialized connective tissue as well as potential mechanisms underlying therapeutic effects of tissue stretch, we developed a carrageenan-induced inflammation model in the low back of a rodent. Induction of inflammation in the lumbar connective tissues resulted in altered gait, increased mechanical sensitivity of the tissues of the low back, and local macrophage infiltration. Mechanical input was then applied to this model as in vivo tissue stretch for 10 minutes twice a day for 12 days. In vivo tissue stretch mitigated the inflammation-induced changes leading to restored stride length and intrastep distance, decreased mechanical sensitivity of the back and reduced macrophage expression in the nonspecialized connective tissues of the low back. This study highlights the need for further investigation into the contribution of connective tissue to low back pain and the need for a better understanding of how interventions involving mechanical stretch could provide maximal therapeutic benefit. This tissue stretch research is relevant to body-based treatments such as yoga or massage, and to some stretch techniques used with physical therapy.     

Monte Carlo evaluation of the effect of inhomogeneities on dose calculation for low energy photons intra-operative radiation therapy in pelvic area

The aim of this study was to evaluate the effect of inhomogeneities on dose calculation for low energy photons intra-operative radiation therapy (IORT) in pelvic area. A GATE Monte Carlo model of the INTRABEAM® was adapted for the study. Simulations were performed in the CT scan of a cadaver considering a homogeneous segmentation (water) and an inhomogeneous segmentation (5 tissues from ICRU44). Measurements were performed in the cadaver using EBT3 Gafchromic® films. Impact of inhomogeneities on dose calculation in cadaver was 6% for soft tissues and greater than 300% for bone tissues. EBT3 measurements showed a better agreement with calculation for inhomogeneous media. However, dose discrepancy in soft tissues led to a sub-millimeter (0.65 mm) shift in the effective point dose in depth. Except for bone tissues, the effect of inhomogeneities on dose calculation for low energy photons intra-operative radiation therapy in pelvic area was not significant for the studied anatomy.     

Translocation of carbohydrates and proline in young grapefruit trees at low temperatures

Girdling and defoliation of fruit-bearing grapefruit (Citrus paradisi Macf.) branches inhibited the accumulation of soluble carbohydrates and proline in fruit tissues during low temperature treatment of trees. These treatments did not inhibit hydrolysis of sucrose to reducing sugars. Flavedo and albedo tissues responded similarly to low temperatures but little or no change occurred in the juice. Therefore, soluble carbohydrates and proline do not appear to interchange between different tissues of the fruit at low temperatures but instead are translocated into the fruit from other parts of the plant. Girdling fruit-bearing branches immediately after low temperature treatments inhibited the accumulation of sucrose in fruit tissues at dehardening temperatures. Also, proline levels decreased rapidly in fruit on girdled branches at dehardening temperatures. This rapid decrease suggests proline may serve as a source for respiratory energy in grapefruit during rapidly changing temperatures that favor active growth and during recovery of citrus from environmental stress.     

Tyrosine hydroxylase assay for detection of low levels of enzyme activity in peripheral tissues

A nonisotopic assay for tyrosine hydroxylase, with optimized signal-to-noise ratios, enables determination of low levels of enzyme activity in peripheral tissues. DOPA produced by the enzyme is measured using HPLC with electrochemical detection. Increased signal-to-noise ratios are obtained by including in the reaction mixture glycerol for reduction of blank values and dihydropteridine reductase and NADPH for regeneration of the tetrahydropteridine cofactor. With this method, tyrosine hydroxylase activity can be detected in as few as 200 PC12 cells and in peripheral tissues at levels as low as 4.5 fmol/min/mg wet weight. The assay permits activity to be assessed in a variety of peripheral tissues.     

The appropriateness of swab cultures for the release of human allograft tissue

Surgeries utilizing human allograft tissues have increased dramatically in recent years. With this increase has come a greater reliance on the use of swab culturing to assess allograft tissues for microbial contamination prior to distribution. In contrast to the typical industrial microbiological uses for swabs, the tissue banking industry has relied on swab cultures as a sterility release method for allograft tissues. It has been reported in the literature that swabs have limitations, both in sensitivity and reproducibility, so their suitability as a final sterility release method was evaluated in this study. Two different swab-culturing systems were evaluated (COPAN, EZ Culturette) using human allograft tissues spiked with low levels of multiple bacterial and fungal microorganisms. The average microbial recoveries for all challenge microorganisms for each tissue type and each swab system were calculated. Percent recoveries for each challenge microorganism were also calculated and reported. The results indicated that both swab systems exhibited low and highly variable recoveries from the seeded allograft tissues. Further analysis indicated there was no statistical difference (∝=0.05) between the two swab systems. It is the recommendation of the authors that swab culturing not be used to assess relatively low levels of microbial contamination on allografts. Instead, alternative validated microbial detection methods with improved sensitivity and reproducibility should be employed and validated for this critical task.     

Low thyroidal T3 in nodular goitrous tissue

Six nodular tissues of non-treated and four of treated patients (suppressive treatment with thyroid hormones from three months to two years until the operation) with nodular non-toxic goitre contained low T3 (less than 1 ug/g w.w.). The results of iodothyronines and thyroglobulin (Tg) were compared with respective tissues containing T3 greater than 1 ug/g w.w. In non-treated patients, nodular tissues with low T3 and very high T4/T3 ratio showed T4 and Tg concentrations not different from the tissues with T3 greater than 1 ug/g w.w. In the goitres with low T3 of treated patients, T4 was also reduced but disproportionately to T3. Microscopically, nodular goitres with low T3 were characterized with gross fibrous infiltration and diffuse haemorrhage which was substantially different from histological findings in nodular goitres with T3 greater than 1 ug/g w.w. High T4/T3 ratio in the tissues with low T3 is similar to increased T4/T3 ratio in paranodular tissues of autonomously functioning adenomas. The results suggest that low T3 and high T4/T3 ratio in nodular goitrous tissue could be due to grossly impaired thyroid function or due to suppressed secretion of TSH.     

The ultrastructure of the outer tissues of cold-stored apple fruits of high and low calcium content in relation to cell breakdown

The outer tissues of Cox's Orange Pippin apples from samples with initially high and low calcium content were examined in the electron microscope following 53, 81 or 99 days in air storage at 3 ^oC. Cell ultrastructure within tissues of both high and low calcium apples was comparatively normal in fruit stored for the shorter period but low calcium fruit showed symptoms of membrane breakdown after 81 days. The greatest structural differences were observed after storage for 99 days when low calcium fruits contained more cells exhibiting breakdown of membranes and organelles than high calcium apples and also showed more severe symptoms of cell wall breakdown.     

抑癌候选基因NDRG2在甲状腺癌组织中的表达及其意义

目的:研究抑癌候选基因NDRG2在人类甲状腺癌组织及其癌旁组织中的表达情况.方法:收集30例甲状腺癌组织及其癌旁组织,提取总RNA,应用半定量RT-PCR方法检测NDRG2 mRNA的表达水平.分别提取30例组织的总蛋白,应用蛋白印迹技术检测其NDRG2的蛋白表达水平.结果:RT-PCR结果显示,30例甲状腺癌组织中,有25例NDRG2的mRNA水平明显降低,蛋白印迹结果显示,30例甲状腺癌组织中发现25例NDRG2的蛋白水平明显下降,与RT-PCR检测结果一致.结论:NDRG2在甲状腺癌组织中呈低表达,提示其可能对甲状腺癌的发生或发展有重要作用影响.     

Effects of salt and osmotic shocks on unadapted and adapted callus lines of tobacco

Growth, viability and proline content of adapted and unadapted calluses of Nicotiana tabacum L. var. Jayasri, affected due to osmotic stresses and particularly to stress-shocks treated with different osmotica like NaCl (ionic-penetrating), mannitol (non-ionic-penetrating) and polyethylene glycol, (PEG) (non-ionic-non penetrating) were studied to evaluate the physiological differences of stress effects. The tissues adapted to a low concentration of NaCl (85 mM) showed low growth with high proline content compared to the tissues adapted to a low concentration of mannitol (165 mM). Proline content was similar in tissues adapted to high concentrations of NaCl (171 mM) and mannitol (329 mM) but growth in the latter case was relatively low. Growth and viability were subsequently correlated with the pattern of retention in or diffusion of proline out of the tissues after shock-treatments. The loss of tissue viability of the adapted calluses was comparatively less than the unadapted callus even after shock-treatments with 1282 mM NaCl and 823 mM mannitol. The former calluses retained the capability of regrowth though at a slow rate. Such adapted tissues also retained more proline. The mannitol-adapted tissues, when shocked with PEG (200 g l-1), showed low viability with more diffusion and a very little retention of proline while, in the unadapted tissue, all the proline was leached out. The results indicated that the effects of different osmotica on plant tissue varied depending upon the physico-chemical nature of the compounds used as stress-inducing-agents, and retention and diffusion of proline was altered when the tissues were shocked with high concentrations of all these compounds. This revised version was published online in June 2006 with corrections to the Cover Date.     

Tissue-dependent regulation of protein tyrosine kinase activity during embryonic development

Protein tyrosine kinase activity was assayed in a variety of chicken tissues during embryonic development and in the adult. In some tissues protein tyrosine kinase activity decreased during embryonic development; however, in other tissues it remained high throughout development, it contrast to the level of protein tyrosine phosphorylation, which decreased during development. The highest levels of tyrosine kinase activity were detected in 17-d embryonic brain although only low levels of protein tyrosine phosphorylation were observed in this tissue. Several alternatives were examined in an effort to determine the mechanism responsible for the low levels of tyrosine phosphorylated proteins in most older embryonic and adult chicken tissues despite the presence of highly active tyrosine kinases. The results show that the regulation of protein tyrosine phosphorylation during embryonic development is complex and varies from tissue to tissue. Furthermore, the results suggest that protein tyrosine phosphatases play an important role in regulating the level of phosphotyrosine in proteins of many older embryonic and adult tissues.     

Lysosome lipid storage disorder in NCTR-BALB/c mice: spleen and lung lysosomes store unesterified cholesterol but differ in their phospholipid composition

A strain derived from a colony of BALB/c mice at the National Center for Toxicological Research, Jefferson, AR, USA (NCTR-BALB/c) suffers from an autosomal recessive disorder characterized by proliferation of secondary lysosomes with accumulation ofunesterified cholesterol in several tissues. The unesterified cholesterol content of spleens and lungs from the affected mice were elevated 8- and 3-fold respectively over age- and sex-matched controls. Postnuclear supernatants of tissue homogenates were fractionated by sucrose density gradient centrifugation and the fractions were analyzed for unesterified cholesterol, protein and marker enzyme activities for lysosomes (N-acetyl-beta-D-glucosaminidase, beta-D-glucuronidase), plasma membrane (alkaline phosphodiesterase I), endoplasmic reticulum (glucose-6-phosphatase) and mitochondria (cytochrome oxidase). The enzyme distribution profile showed that lysosomes of affected tissues floated at low density regions (density 1.05-1.08) of the gradient and contained substantial amount of tissue unesterified cholesterol. These low density lysosomes were purified about 17-fold (58% yield) from spleen and about 6-fold (32% yield) from lungs with minimal contamination by other organelles They were mostly intact as judged by high latency for N-acetyl-beta-D-glucosaminidase activity (70-100%). Lysosomes of control tissues were not found at the low density regions. The distribution profiles for other organelles were similar between affected and control tissues. Phospholipid composition of low density lysosomes were distinctly different from their respective tissue homogenates. Spleen and lung lysosomes were enriched in sphingomyelin and phosphatidylcholine respectively. The results suggest that these lysosomes acquire their low densities due to accumulation of unesterified cholesterol, the retention of which may be aided by sphingomyelin and phosphatidylcholine content of the lysosomes.     

Increased expression of microRNA-9 predicts an unfavorable prognosis in human glioma

microRNA-9 (miR-9) has been found to be upregulated along with tumor progression of gliomas by microarray-based expression profiling, and also be strongly linked to glioblastoma subtypes. However, its prognostic value in glioma is still elusive. miR-9 expression in human gliomas and nonneoplastic brain tissues was measured by real-time quantitative RT-PCR assay. miR-9 expression in glioma tissues was significantly higher than that in corresponding nonneoplastic brain tissues (P < 0.001). The increased expression of miR-9 was more frequently observed in glioma tissues with high WHO grade than those with low WHO grade tissues (P = 0.001). The expression levels of miR-9 in glioma tissues with low Karnofsky performance score (KPS) were also significantly higher than those with high KPS (P = 0.008). Moreover, the overall survival of glioma patients with high miR-9 expression was obviously lower than that with low miR-9 expression (P < 0.001). Multivariate analysis further showed that high miR-9 expression was an independent prognostic factor for overall survival in glioma patients (P = 0.01). More importantly, the subgroup analyses indicated that the overall survival of glioma patients with high WHO grade (III–IV) was significantly worse for high miR-9 expression group than for low miR-9 expression group (P < 0.001), but no significant difference was found for patients with low WHO grade (I–II). These findings suggest for the first time that the increased expression of miR-9 may play an important role in tumor progression in human gliomas. miR-9 might be a useful marker for predicting the clinical outcome of glioma patients, especially for advanced subtypes.     

Antioxidant defense system in tissues of semiaquatic mammals

Activity of antioxidant enzymes (superoxide dismutase and catalase) and low molecular weight antioxidants (tocopherol and retinol) was studied in tissues of 8 semiaquatic mammalian species. In animals from different taxa, the enhancement of antioxidant defense can be achieved either by increased enzymatic activity of tissues or elevated concentration of low molecular weight antioxidants. The level of enzymatic and nonenzymatic (low molecular weight) antioxidants, determined in tissues and organs of diving mammals, is likely to be considered as an integral complex, which has evolved to ensure the greatest efficiency of metabolic systems during adaptation to species-specific habitats.     

Mechanical stimuli and IL-13 interact at integrin adhesion complexes to regulate expression of smooth muscle myosin heavy chain in airway smooth muscle tissue

Airway smooth muscle phenotype may be modulated in response to external stimuli under physiological and pathophysiological conditions. The effect of mechanical forces on airway smooth muscle phenotype were evaluated in vitro by suspending weights of 0.5 or 1 g from the ends of canine tracheal smooth muscle tissues, incubating the weighted tissues for 6 h, and then measuring the expression of the phenotypic marker protein, smooth muscle myosin heavy chain (SmMHC). Incubation of the tissues at a high load significantly increased expression of SmMHC compared with incubation at low load. Incubation of the tissues at a high load also decreased activation of PKB/Akt, as indicated by its phosphorylation at Ser 473. Inhibition of Akt or phosphatidylinositol-3,4,5 triphosphate-kinase increased SmMHC expression in tissues at low load but did not affect SmMHC expression at high load. IL-13 induced a significant increase in Akt activation and suppressed the expression of SmMHC protein at both low and high loads. The role of integrin signaling in mechanotransduction was evaluated by expressing a PINCH (LIM1-2) fragment in the muscle tissues that prevents the membrane localization of the integrin-binding IPP complex (ILK/PINCH/α-parvin), and also by expressing an inactive integrin-linked kinase mutant (ILK S343A) that inhibits endogenous ILK activity. Both mutants inhibited Akt activation and increased expression of SmMHC protein at low load but had no effect at high load. These results suggest that mechanical stress and IL-13 both act through an integrin-mediated signaling pathway to oppositely regulate the expression of phenotypic marker proteins in intact airway smooth muscle tissues. The stimulatory effects of mechanical stress on contractile protein expression oppose the suppression of contractile protein expression mediated by IL-13; thus the imposition of mechanical strain may inhibit changes in airway smooth muscle phenotype induced by inflammatory mediators.     

3-Mercaptopyruvate sulfurtransferase activity in guinea pig and rat tissues

Activities of 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) in guinea pig tissues were determined and compared with those in the corresponding rat tissues. The activities in guinea pig tissues were found to be very low. The activity in the liver was 145.9 mumol pyruvate formed per g of fresh tissue per 15 min. This was 1/50 of the activity in the rat liver. Activities in the kidney and brain were 1/100 of the corresponding rat tissues. Those in the erythrocyte and heart were negligibly low and far less than 1/1000 of these tissues in the rat. Similarities between the guinea pig and patients with beta-mercaptolactate-cysteine disulfiduria are discussed.     

Exposure of fish to high‐intensity sonar does not induce acute pathology

This study investigated immediate effects of intense sound exposure associated with low‐frequency (170–320 Hz) or with mid‐frequency (2·8–3·8 kHz) sonars on caged rainbow trout Oncorhynchus mykiss, channel catfish Ictalurus punctatus and hybrid sunfish Lepomis sp. in Seneca Lake, New York, U.S.A. This study focused on potential effects on inner ear tissues using scanning electron microscopy and on non‐auditory tissues using gross and histopathology. Fishes were exposed to low‐frequency sounds for 324 or 628 s with a received peak signal level of 193 dB re 1 µPa (root mean square, rms) or to mid‐frequency sounds for 15 s with a received peak signal level of 210 dB re 1 µPa (rms). Although a variety of clinical observations from various tissues and organ systems were described, no exposure‐related pathologies were observed. This study represents the first investigation of the effects of high‐intensity sonar on fish tissues in vivo. Data from this study indicate that exposure to low and midfrequency sonars, as described in this report, might not have acute effects on fish tissues.     

The role of antioxidative status in development of the consequences of biological action under low dose and low intensity irradiation

The scale and direction of changes of a number of biophysical, biochemical and physiological parameters in SHK mice (male) tissues were studied depending on their initial values for mice control groups within 1 day after low intensity gamma-irradiation (15 cGy). The reciprocal dependences between the scales of the lipid antioxidative activity (AOA) in brain or the spleen mass changes and their values for mice control groups were found. The external dependences were revealed between the amounts of lipid peroxidation secondary products in spleen, liver and blood plasma of the control mice and a scale of their change after irradiation. The most substantial changes of this parameter were observed in blood plasma and the changes of the phospholipid content within the total lipid composition were found in spleen and blood erythrocytes of the irradiated mice, that is in the lipids of tissues, which had the lowest level of AOA for mice control groups. The experimental data obtained indicate that the initial antioxidant status of animal tissues plays the important role for the development of consequences of the biological action under low dose and low intensity irradiation.