Supplementation with the histone deacetylase inhibitor trichostatin A during in vitro culture of bovine embryos

Summary Trichostatin A (TSA) is a histone deacetylase inhibitor that induces histone hyperacetylation and increases gene expression levels. The aim of the present study was to establish a suitable condition for the use of TSA in in vitro cultures of bovine embryos, and to determine whether TSA would increase blastocyst rates by improvement of chromatin remodelling during embryonic genome activation and by increasing the expression of crucial genes during early development. To test this hypothesis, 8-cell embryos were exposed to four concentrations of TSA for different periods of time to establish adequate protocols. In a second experiment, three experimental groups were selected for the evaluation of embryo quality based on the following parameters: apoptosis, total cell number and blastocyst hatching. TSA promoted embryonic arrest and degeneration at concentrations of 15, 25 and 50 nM. All treated groups presented lower blastocyst rates. Exposure of embryos to 5 nM for 144 h and to 15 nM for 48 h decreased blastocyst hatching. However, the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay (TUNEL) assay revealed similar apoptosis rates and total cell numbers in all groups studied. Although, in the present study, TSA treatment did not improve the parameters studied, the results provided background information on TSA supplementation during in vitro culture of bovine embryos and showed that embryo quality was apparently not affected, despite a decrease in blastocyst rate after exposure to TSA. © Cambridge University Press 2011.

Assessment of central venous catheter-associated infections using semi-quantitative or quantitative culture methods

Semiquantitative (Maki) and quantitative (Brun- Buisson) culture techniques were employed in the diagnosis of catheter-related bloodstream infections (CRBSI) in patients who have a short-term central venous catheter (inserted for 30 days). The diagnosis of CRBSI was based on the results of semiquantitative and quantitative culture of material from the removed catheters. Catheter tips (118) from 100 patients were evaluated by both methods. Semiquantitative analysis revealed 34 catheters (28.8%) colonized by ≥15 colonyforming units (cfu), while quantitative cultures (34 catheters, 28.8%) showed the growth of ≥103 cfu/mL. Bacteremia was confirmed in four patients by isolating microorganisms of identical species from both catheters and blood samples. Using the semiquantitative culture technique on short-term central venous catheter tips, we have shown that with a cut-off level of ≥15 cfu, the technique had 100.0% sensitivity, specificity of 68.4%, 25.0% positive predictive value (PPV) and 100.0% negative predictive value (NPV), efficiency of 71.4% and a prevalence of 9.5%. The quantitative method, with a cut-off limit of ≥103 cfu/mL, gave identical values: the sensitivity was 100.0%, specificity 68.4%, positive predictive value (PPV) 25.0%, negative predictive value (NPV) 100.0%, efficiency 71.4% and prevalence 9.5%. We concluded that the semiquantitative and quantitative culture methods, evaluated in parallel, for the first time in Brazil, have similar sensitivity and specificity. Keywords: central venous catheter; semi-quantitative culture; quantitative culture; catheter-related bacteremia.

Axenic cell culture of the seagrass Cymodocea nodosa from cotyledonary tissue

[EN] Plant Tissue Culture, also called “micropropagation”, is the propagation of plants from different tissues (or explants) in a shorter time than conventional propagation, making use of the ability that many plant cells have to regenerate a whole plant (totipotency).There are two alternative mechanisms by which an explant can regenerate an entire plant, namely organogenesis and somatic embryogenesis. Since the last decades, the number of higher terrestrial plants species from which these techniques have been successfully applied has continually increased. However, few attempts have been carried out in marine plants. Previous seagrasses authors have focused their studies on i) vegetative propagation of rhizome fragments as explants in Ruppia maritima, Halophila engelmannii, Cymodocea nodosa and Posidonia oceanica; ii) culture of meristems in Heterozostera tasmanica, C. nodosa or P. oceanica; and iii) culture of germinated seeds on aseptic conditions, in Thalassia testudinum, H. ovalis, P. coriacea, P. oceanica, and H. decipiens. All these studies determine the most adequate culture medium for each species (seawater, nutrients, vitamins, carbon sources, etc...), often supplemented with different plant growth regulators and the necessary conditions for the culture maintenance, such as light and temperature. On the other hand, several studies have previously established protocols for cell or protoplast isolation in the species Zostera marina, Z. muelleri, P. oceanica, and C. nodosa, using shoots collected from natural meadows as original vegetal source, but further cell growth was never accomplished. Due to the absence of somatic embryogenesis or organogenetic studies in seagrasses we wonder: IS THE SUCCESSFUL APPLICATION OF TISSUE CULTURE TECHNIQUES POSSIBLE IN SEAGRASSES?

Time course modifications in organotypic culture of human neuroretina

The purpose of this study was to characterize organ culture of human neuroretina and to establish survival and early degeneration patterns of neural and glial cells. Sixteen neuroretina explants were prepared from 2 postmortem eyes of 2 individuals. Four explants were used as fresh retina controls, and 12 were evaluated at 3, 6, and 9 days of culture. Neuroretina explants (5 × 5 mm) were cultured in Transwell® dishes with the photoreceptor layer facing the supporting membrane. Culture medium (Neurobasal A-based) was maintained in contact with the membrane beneath the explant. Cryostat and ultrathin sections were prepared for immunohistochemistry and electron microscopy. Neuroretinal modifications were evaluated after toluidine blue staining and after immunostaining for neuronal and glial cell markers. Ultrastructural changes were analyzed by electron microscopy. From 0 to 9 days in culture, there was progressive retinal degeneration, including early pyknosis of photoreceptor nuclei, cellular vacuolization in the ganglion cell layer, decrease of both plexiform layer thicknesses, disruption and truncation of photoreceptor outer segments (OS), and marked reduction in the number of nuclei at both nuclear layers where the cells were less densely packed. At 3 days there was swelling of cone OS with impairment of pedicles, loss of axons and dendrites of horizontal and rod bipolar cells that stained for calbindin (CB) and protein kinase C (PKC-α), respectively. After 9 days, horizontal cells were pyknotic and without terminal tips. There were similar degenerative processes in the outer plexiform layer for rod bipolar cells and loss of axon terminal lateral varicosities in the inner plexiform layer. Glial fibrillary acidic protein (GFAP) staining did not reveal a dramatic increase of gliosis in Müller cells. However, some Müller cells were CB immunoreactive at 6 days of culture. Over 9 days of culture, human neuroretina explants underwent morphological changes in photoreceptors, particularly the OS and axon terminals, and in postsynaptic horizontal and bipolar cells. These early changes, not previously described in cultured human samples, reproduce some celullar modifications after retinal damage. Thus, this model may be suitable to evaluate therapeutic agents during retinal degeneration processes.

Exploring plant tissue culture to improve the production of phenolic compounds: A review

Plant tissue and organ culture has been extensively used from the beginning of the XX century for the study and comprehension of some primary biological mechanisms such as morphogenesis. However, with the increasing demand of the market for novel products derived from plants, in vitro culture became a reliable technique for the mass production of plant material. Moreover, the potential to use this technique for the production of some bioactive compounds, such as phenolic compounds, is immense since it allows the manipulation of the biosynthetic routes to increase the production and accumulation of specific compounds. This work intends to make a brief historical review of in vitro culture, highlighting its use for the production of bioactive compounds. Also, emphasizes the importance of phenolic compounds for the consumer as well reviews the metabolic pathways involved in its production in plant cells. Furthermore, it was carried out a comprehensive study on the work developed for the production of plant phenolic compounds in in vitro cultures, as well as on the type of elicitors used to increase of the same production; also a brief highlighting of the phenolic compounds which serve as elicitors. There are numerous reports directed to the production of phenolic extracts in in vitro plant cultures, however there is a lack in the production of individual phenolic compounds mainly due to the complexity of the biosynthetic routes and extraction procedures. Elicitation procedures are often used to increase the production of phenolics, archieving in most cases higher yields than in non-elicitated cultures. The increasing production of bioactive phenolic extracts/compounds allows for their further applicability, namely in the industry of functional foods or in pharmaceutical/medical fields.

An Efficient Protocol For The Generation Of Monocyte Derived Dendritic Cells Using Serum-free Media For Clinical Applications In Post Remission Aml Patients.

Protocols for the generation of dendritic cells (DCs) using serum as a supplementation of culture media leads to reactions due to animal proteins and disease transmissions. Several types of serum-free media (SFM), based on good manufacture practices (GMP), have recently been used and seem to be a viable option. The aim of this study was to evaluate the results of the differentiation, maturation, and function of DCs from Acute Myeloid Leukemia patients (AML), generated in SFM and medium supplemented with autologous serum (AS). DCs were analyzed by phenotype characteristics, viability, and functionality. The results showed the possibility of generating viable DCs in all the conditions tested. In patients, the X-VIVO 15 medium was more efficient than the other media tested in the generation of DCs producing IL-12p70 (p=0.05). Moreover, the presence of AS led to a significant increase of IL-10 by DCs as compared with CellGro (p=0.05) and X-Vivo15 (p=0.05) media, both in patients and donors. We concluded that SFM was efficient in the production of DCs for immunotherapy in AML patients. However, the use of AS appears to interfere with the functional capacity of the generated DCs.

Does The Reciproc File Remove Root Canal Bacteria And Endotoxins As Effectively As Multifile Rotary Systems?

To evaluate the effectiveness of Reciproc for the removal of cultivable bacteria and endotoxins from root canals in comparison with multifile rotary systems. The root canals of forty human single-rooted mandibular pre-molars were contaminated with an Escherichia coli suspension for 21 days and randomly assigned to four groups according to the instrumentation system: GI - Reciproc (VDW); GII - Mtwo (VDW); GIII - ProTaper Universal (Dentsply Maillefer); and GIV -FKG Race(™) (FKG Dentaire) (n = 10 per group). Bacterial and endotoxin samples were taken with a sterile/apyrogenic paper point before (s1) and after instrumentation (s2). Culture techniques determined the colony-forming units (CFU) and the Limulus Amebocyte Lysate assay was used for endotoxin quantification. Results were submitted to paired t-test and anova. At s1, bacteria and endotoxins were recovered in 100% of the root canals investigated (40/40). After instrumentation, all systems were associated with a highly significant reduction of the bacterial load and endotoxin levels, respectively: GI - Reciproc (99.34% and 91.69%); GII - Mtwo (99.86% and 83.11%); GIII - ProTaper (99.93% and 78.56%) and GIV - FKG Race(™) (99.99% and 82.52%) (P < 0.001). No statistical difference were found amongst the instrumentation systems regarding bacteria and endotoxin removal (P > 0.01). The reciprocating single file, Reciproc, was as effective as the multifile rotary systems for the removal of bacteria and endotoxins from root canals.

Clinical Influence Of Different Intracanal Medications On Th1-type And Th2-type Cytokine Responses In Apical Periodontitis.

This clinical study assessed the influence of different intracanal medications on Th1-type and Th2-type cytokine responses in apical periodontitis and monitored the levels of bacteria from primarily infection during endodontic procedures. Thirty primarily infected teeth were randomly divided into 3 groups according to the medication selected: chlorhexidine (CHX), 2% CHX gel; Ca(OH)2/SSL, Ca(OH)2 + SSL; and Ca(OH)2/CHX, Ca(OH)2 + 2% CHX gel (all, n = 10). Bacterial sample was collected from root canals, and the interstitial fluid was sampled from lesions. Culture techniques were used to determine bacterial counts (colony-forming units/mL). Th1 (tumor necrosis factor-α, interferon-γ, and interleukin [IL]-2) and Th2 cytokines (IL-4, IL-5, and IL-13) were measured by enzyme-linked immunosorbent assay. All intracanal medication protocols were effective in reducing the bacterial load from root canals (all P < .05) and lowering the levels of Th1-type cytokines in apical lesions (all P < .05), with no differences between them (P > .05). Both Ca(OH)2 treatment protocols significantly increased the levels of Th2-type cytokines (P < .05), with no differences between them (P > .05). Thus, chlorhexidine medication showed the lowest effectiveness in increasing the levels of Th2-type cytokine. After treatment, regardless of the type of medication, the linear regression analysis indicated the down-regulation of Th2-type cytokines by Th1-type cytokines. All intracanal medication protocols were effective in reducing bacterial load and lowering the levels of Th1-type cytokines. Thus, the use of Ca(OH)2 medications contributed to the increase in the Th2-type cytokine response in apical periodontitis.

Direct regeneration of protocorm-like bodies (PLBs) from leaf apices of Oncidium flexuosum Sims (Orchidaceae)

The present study describes the direct regeneration of protocorm-like bodies (PLBs) in leaf explants of the tropical species Oncidium flexuosum. The explants were inoculated in a solid, modified Murashige and Skoog (MS) medium with different concentrations of the growth regulator thidiazuron (TDZ) and with or without 2,4-dichlorophenoxyacetic acid (2,4-D) and naphthalene acetic acid (NAA), and kept away from light or in a 16-h photoperiod. The presence of auxins, 2,4-D, and NAA inhibited the formation of PLBs. The highest frequency of explants that regenerated PLBs (80%) was obtained when they were maintained in a culture medium containing 1.5 mu M TDZ under dark conditions. In the same culture medium but under a 16-h photoperiod, 95% of the leaf explants presented necrosis. Therefore, darkness was crucial for the regeneration of PLBs in O. flexuosum leaf explants, which is in disagreement with the literature. PLBs developed from the division of epidermal and subepidermal cells mainly on the adaxial side of the apex region of the explant. Plants with well-developed leaves and roots grew after the PLBs were transferred to growth regulator-free medium under a 16-h photoperiod.

Antioxidant response of Nicotiana tabacum cv. Bright Yellow 2 cells to cadmium and nickel stress

Plant cell cultures are a suitable model system for investigation of the physiological mechanisms of tolerance to environmental stress. We have determined the effects of Cd (0.1 and 0.2 mM CdCl(2)) and Ni (0.075 and 0.75 mM NiCl(2)) on Nicotiana tabacum L. cv. Bright Yellow (TBY-2) cell suspension cultures over a 72-h period. Inhibition of growth, loss of cell viability and lipid peroxidation occurred, in general, only when the TBY-2 cells were grown at 0.2 mM CdCl(2) and at 0.75 mM NiCl(2). At 0.1 mM CdCl(2), a significant increase in growth was determined at the end of the experiment. Increases in the activities of all of the four enzymatic antioxidant defence systems tested, were induced by the two concentrations of Cd and Ni, but at different times during the period of metal exposure. Overall, the cellular antioxidant responses to Cd and Ni were similar and were apparently sufficient to avoid oxidative stress at the lower concentrations of Cd and Ni. The activities of glutathione reductase and glutathione S-transferase increased early but transiently, whereas the activities of catalase and guaiacol peroxidase increased in the latter half of the experimental period. Therefore it is likely that the metabolism of reduced glutathione was enhanced during the initial onset of the stress, while catalase and guaiacol-type peroxidase appeared to play a more important role in the antioxidant response once the stress became severe.

Tamoxifen inhibits transforming growth factor-alpha gene expression in human breast carcinoma samples treated with triiodothyronine

Objectives: To examine the effects of triiodothyronine (T(3)), 17 beta-estradiol (E(2)), and tamoxifen (TAM) on transforming growth factor (TGF)-alpha gene expression in primary breast cancer cell cultures and interactions between the different treatments. Methods and results: Patients included in the study (no.=12) had been newly diagnosed with breast cancer. Fresh human breast carcinoma tissue was cut into 0.3-mm slices. These slices were placed in six 35-mm dishes on 2-ml organ culture medium. Dishes received the following treatments: dish 1: ethanol; dish 2: T(3); dish 3: T(3)+TAM; dish 4: TAM; dish 5: E(2); dish 6: E(2)+TAM. TGF-alpha mRNA content was normalized to glyceraldehyde-3-phosphate dehydrogenase mRNA levels. All tissues included in this study were positive for estrogen receptor (ER) and thyroid hormone receptor expression. Treatment with T(3) for 48 h significantly increased TGF-alpha mRNA levels compared to controls (15-fold), and concomitant treatment with TAM reduced expression to 3.4-fold compared to controls. When only TAM was added to the culture medium, TGF-alpha mRNA expression increased 5.3-fold, significantly higher than with all other treatment modalities. Conclusion: We demonstrate that TGF-alpha mRNA expression is more efficiently upregulated by T(3) than E(2). Concomitant treatment with TAM had a mitigating effect on the T(3) effect, while E(2) induced TGF-alpha upregulation. Our findings show some similarities between primary culture and breast cancer cell lines, but also some important differences: a) induction of TGF-alpha, a mitogenic protein, by TAM; b) a differential effect of TAM that may depend on relative expression of ER alpha and beta; and c) supraphysiological doses of T3 may induce mitogenic signals in breast cancer tissue under conditions of low circulating E(2).. Endocrinol. Invest. 31: 1047-1051, 2008) (c) 2008, Editrice Kurtis

G2 phase cell cycle arrest in human skin following UV irradiation

The contribution of the short wavelength ultraviolet (UV) component of sunlight to the aetiology of skin cancer has been widely acknowledged, although its direct contribution to tumour initiation or progression is still poorly understood. The loss of normal cell cycle controls, particularly checkpoint controls, are a common feature of cancer. UV radiation causes both GI and G2 phase checkpoint arrest in vitro cultured cells. In this study we have investigated the cell cycle responses to suberythemal doses of UV on skin. We have utilized short-term whole organ skin cultures, and multi parameter immunohistochemical and biochemical analysis to demonstrate that basal and suprabasal layer melanocytes and keratinocytes undergo a G2 phase cell cycle arrest for up to 48 h following irradiation. The arrest is associated with increased p16 expression but no apparent p53 involvement. This type of organ culture provides a very useful model system, combining the ease of in vitro manipulation with the ability to perform detailed molecular analysis in a normal tissue environment.

An immunofluorescence test for diagnosis of ophthalmic herpes in a mouse corneal model

Herpes simplex virus type 1 (HSV-1) ophthalmic disease is the most common cause of corneal blindness in humans world-wide. Current culture techniques for HSV take several days and commercially available HSV laboratory based diagnostic techniques vary in sensitivity. Our study was conducted to evaluate the use of a quicker and simpler method to herpes ophthalmic diagnosis. Corneal smears were made by firm imprints of infected mouse eyes to glass slides, after smears were fixated with cold acetone, and an indirect immunofluorescence (IIF) method was performed using monoclonal antibodies in a murine model of ophthalmic herpes. Eye swabs from infected mice were inoculated in Vero cells for virus isolation. Cytology and histology of the eye were also performed, using hematoxylin-eosin routine. Mouse eyes were examined by slit-lamp biomicroscopy for evidence of herpetic disease at various times postinoculation. We made a comparative evaluation of sensitivity, specificity and speed of methods for laboratory detection of HSV. Our results indicate that this IIF method is quick, sensitive, specific and can be useful in the diagnosis of ophthalmic herpes as demonstrated in an animal model.

Cells with neuronal characteristics differentiate and persist for one year in rat optic nerve explants cultures.

Evidence concerning the presence or absence of common neuronglia lineages in the postnatal mammalian central nervous system is still a matter of speculation. We address this problem using optic nerve explants, which show an extremely long survival in culture. Morphological, immunocytochemical and immunochemical methods were applied. The results obtained from in vitro tissue were compared with optic nerves (ONs) and whole-brain samples from animals of different ages. Newborn rat ONs represented the starting material of our tissue culture; they are composed of unmyelinated axons, astrocytes and progenitor cells but devoid of neuronal cell bodies. At this age, Western blots of ONs were positively stained by neurofilament and synapsin I specific antibodies. These bands increased in intensity during postnatal in situ development. In explant cultures, the glia cells reach a stage of functional differentiation and they maintain, together with undifferentiated cells, a complex histotypic organization. After 6 days in vitro, neurofilaments and synapsin I could not be detected on immunoblots, indicating that 1) axonal degeneration was completed, and 2) neuronal somata were absent at the time. Surprisingly, after about 4-5 weeks in culture, a new cell type appeared, which showed characteristics typical of neurons. After 406 days in vitro, neurofilaments and synapsin I were unequivocally detectable on Western blots. Furthermore, both immunocytochemical staining and light and electron microscopic examinations corroborated the presence of this earlier-observed cell type. These in vitro results clearly show the high developmental plasticity of ON progenitor cells, even late in development. The existence of a common neuron-glia precursor, which never gives rise to neurons in situ, is suggested.

An optimized method for establishing high purity murine CD8+ T cell cultures.

Establishing CD8(+) T cell cultures has been empirical and the published methods have been largely individual laboratory based. In this study, we optimized culturing conditions and show that IL-2 concentration is the most critical factor for the success of establishing CD8(+) T cell cultures. High IL-2 concentration encouraged T cells to non-specifically proliferate, express a B cell marker, B220, and undergo apoptosis. These cells also lose typical irregular T cell morphology and are incapable of sustaining long-term cultures. Using tetramer and intracellular cytokine assessments, we further demonstrated that many antigen-specific T cells have been rendered nonfunctional when expanded under high IL-2 concentration. When IL-2 is used in the correct range, B220-mediated cell depletion greatly enhanced the success rate of such T cell cultures.