Environmental impact of Holstein Friesian and 3-breed crossbred dairy cows using a Life Cycle Assessment approach applied to individual animals.

This study aimed to set up a Life Cycle Assessment (LCA) approach at level of individual animals to assess the effects of a 3-breed crossbreeding program on the environmental impact of cows. It involved 564 cows, 279 purebred Holstein (HO) and 285 crossbreds (CR), originated from a 3-breed crossbreeding program based on the rotational use of Viking Red, Montebèliarde and HO sires and kept in 2 dairy herds of northern Italy (224 and 340 cows/herd, respectively). The reference unit of the LCA model was the lifetime of cows, from the birth to culling or death. Data were collected at different levels: individual animal-based data referred to the whole life (birth, calving, dry, cull or death dates, and milk production); individual test-date collection of body measures and BCS, used to predict body weight and to estimate energy requirements; common farm-based data concerning herd management (diets composition, and materials used). Data were used to compute: dry matter intake, milk and milk components production, gross income (GI) and income over feed costs (IOFC) pertaining to the lifespan of cows. An individual LCA-derived approach was set up to compute global warming potential (GWP), acidification and eutrophication potential (AP and EP, respectively), and land occupation (LO), which have been associated with different functional units (cow in her whole life or per d of life; kg of milk fat plus protein, and € of GI and of IOFC produced in the herd life). Data were analyzed using a generalized linear model including the fixed effects of genetic group (CR vs HO), farm and their interaction (genetic group x farm). Compared with HO, CR cows completed more lactations (+12%), had earlier first calving (-2 weeks), yielded more fat plus protein in milk both in the lifespan (+8%) and per d of life (+4%). Concerning the environmental impact, when compared with HO herd mates, CR cows had nominal greater emissions per cow in the whole life, similar emissions per d of life and nearly 3% lower GWP, AP and EP per kg of fat plus protein yielded in lifespan. Income over feed costs per unit of emission tended to be nearly 4% greater in CR compared with HO cows. Also the use of land tended to be lower in CR compared with HO in most indicators considered. In conclusion, LCA could be adapted to represent individual animals. Moreover, managing dairy cows according to a 3-breed rotational crossbreeding scheme may be regarded as a strategy that can contribute to mitigate the emissions and to improve the environmental impact of dairy operations.

Optical polarization from colliding stellar stream shocks in a tidal disruption event.

A tidal disruption event (TDE) occurs when a supermassive black hole rips apart a passing star. Part of the stellar material falls toward the black hole, forming an accretion disk that in some cases launches a relativistic jet. We performed optical polarimetry observations of a TDE, AT 2020mot. We find a peak linear polarization degree of 25 ± 4%, consistent with highly polarized synchrotron radiation, as is typically observed from relativistic jets. However, our radio observations, taken up to 8 months after the optical peak, do not detect the corresponding radio emission expected from a relativistic jet. We suggest that the linearly polarized optical emission instead arises from shocks that occur during accretion disk formation, as the stream of stellar material collides with itself.

Cull cow carcass traits and risk of culling of Holstein cows and 3-breed rotational crossbred cows from Viking Red, Montbéliarde, and Holstein bulls.

Culled dairy cows represent a considerable source of meat production, but their carcasses may vary greatly in quality because of the wide variation in the age, stage of lactation, breed, body condition, and other characteristics of the cows at slaughter. However, the effect of crossbreeding on the value of culled cows has so far received little investigation. The aim of this observational study was to compare a range of carcass attributes of cull cows from 3-breed rotational crossbreeding using Viking Red, Montbéliarde (MO), and Holstein (HO) bulls with those of HO purebred cows. Data on 1,814 dairy cows were collected. Cows were reared together in one herd and slaughtered in 4 slaughterhouses. The carcass weight, fleshiness, and fatness scores, the total value, and the price (€/kg) of each cow carcass were recorded. The culling of a few cows in the sample (n = 86) was classified by the farm manager as "urgent" following a diagnosis of injury or sickness, and this information was recorded. Carcass traits were analyzed with a mixed model which included the fixed effects of parity, days in milk, genetic group (purebred HO, 787 cows, and crossbred cows, classified according to the breed of sire within crossbreds, with 309, 428, and 290 cows sired by Viking Red, MO, and HO bulls, respectively), and interactions, and the random effects of month × year of the date of slaughter, and slaughterhouse. Logistic regression was used to investigate the association of parity, days in milk and purebred or crossbred origin with unplanned, "urgent" culling compared with regular culling. Average carcass weight across genetic groups was 297 ± 65 kg, average price €2.03 ± 0.53/kg, and average value €631 ± 269. Compared with HO, crossbred carcasses were 7 to 12% heavier depending on the breed of sire, were graded + 0.12 to + 0.28 units higher for fleshiness and + 0.26 to + 0.30 units higher for fatness, and fetched an 8 to 11% higher price. As a consequence, compared with purebred HO, carcasses from crossbreds had 15 to 24% higher value (€84 to €133 more per cow), with crossbred cows sired by MO showing the greatest values. Moreover, compared with the HO cows, the crossbred cows had a 37% lower risk of being urgently removed from the herd, which raises welfare concerns and may reduce the salvage value of cull cows. Because cull cows represent a supplemental source of income for dairy farmers, the greater overall value of crossbred cull cows should be taken into account in evaluating the economic effectiveness of crossbreeding schemes.

A kilonova as the electromagnetic counterpart to a gravitational-wave source.

Gravitational waves were discovered with the detection of binary black-hole mergers and they should also be detectable from lower-mass neutron-star mergers. These are predicted to eject material rich in heavy radioactive isotopes that can power an electromagnetic signal. This signal is luminous at optical and infrared wavelengths and is called a kilonova. The gravitational-wave source GW170817 arose from a binary neutron-star merger in the nearby Universe with a relatively well confined sky position and distance estimate. Here we report observations and physical modelling of a rapidly fading electromagnetic transient in the galaxy NGC 4993, which is spatially coincident with GW170817 and with a weak, short γ-ray burst. The transient has physical parameters that broadly match the theoretical predictions of blue kilonovae from neutron-star mergers. The emitted electromagnetic radiation can be explained with an ejected mass of 0.04 ± 0.01 solar masses, with an opacity of less than 0.5 square centimetres per gram, at a velocity of 0.2 ± 0.1 times light speed. The power source is constrained to have a power-law slope of -1.2 ± 0.3, consistent with radioactive powering from r-process nuclides. (The r-process is a series of neutron capture reactions that synthesise many of the elements heavier than iron.) We identify line features in the spectra that are consistent with light r-process elements (atomic masses of 90-140). As it fades, the transient rapidly becomes red, and a higher-opacity, lanthanide-rich ejecta component may contribute to the emission. This indicates that neutron-star mergers produce gravitational waves and radioactively powered kilonovae, and are a nucleosynthetic source of the r-process elements.

Early symptoms and long-term clinical outcomes of distal limb's cutaneous arterio-venous malformations: a retrospective multicentre study of 19 adult patients.

INTRODUCTION: Cutaneous arterio-venous malformations (AVM) are high-flow vascular malformations made up of a direct link between arteries and veins without intermediary capillary space. 'Distal limb's AVM', which mean involving hands or feet, are rare and their functional prognosis is often poor. Little is known about their early clinical symptoms. The objectives of this study were to identify early clinical symptoms of distal limb's cutaneous AVMs and to determine their long-term clinical outcome. METHODS: A retrospective study was carried out including adult patients who had distal limb's AVM, who were followed up between January 2000 and November 2013 in two regional tertiary care centres. The information was collected from patients' clinical records and completed by a structured telephone questionnaire. RESULTS: Nineteen patients were included in the study: four (21%) with foot AVM and 15 (79%) with hand AVM. The first clinical symptoms were as follows: swelling (47%), pain (47%), one or several venous dilatations (37%) and rarely abnormal skin colour, hyperthermia and pulsating sensation. The median diagnosis delay was 9 years after the onset of first manifestations. Amongst the 17 patients who underwent a treatment, 53% had embolotherapy session(s), 12% surgery and 35% had both. After an average follow-up of 57.6 months, 31% of the 13 patients contacted who were receiving treatment were in complete remission; 31% had partial remission; 15% had relapse after initial improvement and 23% had treatment failure. Overall, 74% of patients had a serious development of the AVM: 37% had digital or hand amputation, and 42% remained symptomatic and/or unstable. CONCLUSION: This study suggests that initial manifestations of distal limb's AVMs are discreet and non-specific, leading to a diagnosis delay of about 10 years, with poor prognosis. Doctors should evoke the diagnosis earlier, when these symptoms are shown: pain and/or swelling, sometimes with a large vein.

Reaction norms for the study of genotype-environment interaction for growth and indicator traits of sexual precocity in Nellore cattle.

The objective of this study was to quantify the magnitude of genotype-environment interaction (GxE) effects on age at first calving (AFC), scrotal circumference (SC), and yearling weight (YW) in Nellore cattle using reaction norms. For the study, 89,152 weight records of female and male Nellore animals obtained at yearling age were used. Genetic parameters were estimated with a single-trait random-regression model using Legendre polynomials as base functions. The heritability estimates were of low to medium magnitude for AFC (0.05 to 0.47) and of medium to high magnitude for SC (0.32 to 0.51) and YW (0.13 to 0.72), and increased as the environmental gradient became more favorable. The genetic correlation estimates ranged from 0.25 to 1.0 for AFC, from 0.71 to 1.0 for SC, and from 0.42 to 1.0 for YW. High Spearman correlation coefficients were obtained for the three traits, ranging from 0.97 to 0.99. The reaction norms along the environmental gradient of 10 sires each with the highest or lowest breeding value for YW predicted by single-trait analysis demonstrated more plastic phenotypes for YW and more robust phenotypes for SC. The effect of GxE was most important for YW and AFC with respect to SC. When animals are selected for higher SC or YW or lower AFC, considering or not the GxE effect, it is expected that the same animals will be selected. The reaction norms obtained based on sire breeding values along the environmental gradient showed that animals with extreme breeding values respond differently as environmental conditions improve.

Genotype × environment interaction for age at first calving, scrotal circumference, and yearling weight in Nellore cattle using reaction norms in multitrait random regression models.

The aim of this study was to evaluate the effect of genotype × environment interaction (G×E) on age at first calving (AFC), scrotal circumference (SC), and yearling weight (YW) and to estimate genetic correlations between these traits in Nellore cattle using reaction norms in multitrait random regression models. In this study, 28,871, 41,386, and 89,152 records of Nellore cattle for AFC, SC, and YW, respectively, were used. The data were obtained from farms located in the north, northeast, midwest, and southeast regions of Brazil that participate in the DeltaGen Breeding Program. Environmental levels were defined as a function of contemporary groups, that is, animals born in the same herd and year, from the same management group (from birth to yearling), and of the same sex. Postweaning weight gain was used as a criterion to evaluate the environmental conditions for all traits. For reaction norm analyses, residual variances were modeled with homogeneous and heterogeneous classes. The model for SC and YW included the fixed effects of contemporary group and age of the animal as a covariate as well as random direct additive genetic and residual effects. The same model, excluding the covariate age of the animal, was used for AFC. The heritability estimates were low to high for AFC (0.09 to 0.50), high for SC (0.51 to 0.67), and moderate to high for YW (0.33 to 0.71). The genetic correlations (within each trait) along the environmental levels varied from -0.27 to 1.0 for AFC, from 0.73 to 1.0 for SC, and from 0.26 to 1.0 for YW. The genetic correlations between different traits in different environments varied from -0.14 to -0.60 between AFC and SC, from -0.05 to -0.32 between AFC and YW, and from -0.05 to 0.72 between YW and SC. The genetic correlations have had different magnitudes for AFC, SC, and YW, which could indicate the presence of G×E. The present results should support researchers and farmers in defining selection criteria to improve growth traits and sexual precocity. Our results suggest that animals for breeding have to be selected in the same environment and management conditions as their progeny will be reared.

[Localized inflammatory alopecia of the scalp: an unusual presentation of tularemia]. / Plaque inflammatoire alopéciante du cuir chevelu : mode de révélation atypique d'une tularémie.

BACKGROUND: Tularaemia is a rare arthropod-borne zoonotic infection with 20 to 70 new cases being seen each year in France. Cutaneous ulceration and regional lymphadenopathy are the classical dermatological signs. Diagnosis of atypical forms is more complex. OBSERVATION: A 48-year-old woman was admitted for an erythematous papular alopecic lesion of the scalp accompanied by fever, chills and cervical lymphadenopathy. Initial antibiotic therapy for 20 days with amoxicillin clavulanate was ineffective. The patient's history included an episode of hunting in the forest three days before the onset of signs. Finally, serology led to the diagnosis of tularaemia. Combined levofloxacin and doxycycline resulted in regression of the scalp lesion and lymph node disorder. DISCUSSION: The existence of alopecia and location on the scalp did not initially suggest a diagnosis of tularaemia to us. The clinical presentation was highly suggestive of impetigo with satellite lymphadenopathies. However, resistance to antibiotics and the absence of inflammation militated against this diagnosis, and other possible diagnoses such as a tick-borne lymphadenopathy (TIBOLA), borreliosis and tularaemia were discussed. The most common clinical presentation of tularaemia is ulceroglandular tularaemia, which predominates in 80% of cases. The inoculation chancre at the point of initial infection is most often located in the upper limbs. CONCLUSION: An inflammatory plaque on the scalp with alopecia may reveal tularaemia, a potentially fatal disease resulting from inoculation.

Mast cell/IL-4 control of Francisella tularensis replication and host cell death is associated with increased ATP production and phagosomal acidification.

Mast cells are now recognized as effective modulators of innate immunity. We recently reported that mast cells and secreted interleukin-4 (IL-4) effectively control intramacrophage replication of Francisella tularensis Live Vaccine Strain (LVS), and that mice deficient in mast cells or IL-4 receptor (IL-4R(-/-)) exhibit greater susceptibility to pulmonary challenge. In this study, we further evaluated the mechanism(s) by which mast cells/IL-4 control intramacrophage bacterial replication and host cell death, and found that IL-4R(-/-) mice exhibited significantly greater induction of active caspase-3 within lung macrophages than wild-type animals following intranasal challenge with either LVS or the human virulent type A strain SCHU S4. Treatment of LVS-infected bone-marrow-derived macrophages with a pancaspase inhibitor (zVAD) did not alter bacterial replication, but minimized active caspase-3 and other markers (Annexin V and propidium iodide) of cell death, whereas treatment with both rIL-4 and zVAD resulted in concomitant reduction of both parameters, suggesting that inhibition of bacterial replication by IL-4 was independent of caspase activation. Interestingly, IL-4-treated infected macrophages exhibited significantly increased ATP production and phagolysosomal acidification, as well as enhanced mannose receptor upregulation and increased internalization with acidification, which correlated with observations in mast cell-macrophage co-cultures, with resultant decreases in F. tularensis replication.

Inhibition of HIV-1 in cell culture by oligonucleotide-loaded nanoparticles.

PURPOSE: To investigate the potential use of polymeric nanoparticles for the delivery of antisense oligonucleotides in HIV-1-infected cell cultures. METHODS: Phosphorothioate oligonucleotides were encapsulated into poly (D,L-lactic acid) nanoparticles. Two models of infected cells were used to test the ability of nanoparticles to deliver them. HeLa P4-2 CD4+ cells, stably transfected with the beta-galactosidase reporter gene, were first used to evaluate the activity of the oligonucleotides on a single-round infection cycle. The acutely infected lymphoid CEM cells were then used to evaluate the inhibition of the viral production of HIV-1 by the oligonucleotides. RESULTS: The addition to infected CEM cells of nanoparticles containing gag antisense oligonucleotides in the nanomolar range led to strong inhibition of the viral production in a concentration-dependent manner. Similar results were previously observed in HeLa P4-2 CD4+ cells. Nanoparticle-entrapped random-order gag oligonucleotides had similar effects on reverse transcription. However, the reverse transcriptase activity of infected cells treated with nanomolar concentrations of free antisense and random oligonucleotides was not affected. CONCLUSIONS: These results suggest that poly (D,L-lactic acid) nanoparticles may have great potential as an efficient delivery system for oligonucleotides in HIV natural target cells, i.e., lymphocytic cells.

Comparison of two methods of encapsulation of an oligonucleotide into poly(D,L-lactic acid) particles.

The aim of this study was to compare two methods to encapsulate a 25-mer-phosphorothioate oligonucleotide (ODN) into poly(D,L-lactic acid) (PLA) particles. Antisense oligonucleotides belong to a new therapeutic class especially attractive for the treatment of cancers and viral diseases. The development of these new drugs suffers, however, from poor stability in biological media and very low cellular uptake. Polymeric particulate systems display interesting features for ODN delivery. ODN are highly hydrophilic and most encapsulation methods are inappropriate for such molecules. Using poly(D,L-lactide) polymer, two methods of encapsulation were compared. First, a double emulsion technique was used to prepare nano- and microparticles. Secondly, the ODN was combined with a quaternary ammonium, the cethyltrimethyl-ammonium bromide (CTAB), to enhance the hydrophobicity of the molecule before entrapment by the emulsification-diffusion method. Both methods led to the formation of individualized and spherical particles loaded with a significant amount of ODN. Similar entrapment efficiencies were obtained for the nanoparticles prepared by both methods (approx. 27% of the theoretical loading) whereas 45% of entrapment efficiency was observed for the microparticles. Seventy five percent of the ODN were released in 60 min with the particles prepared by the emulsification-diffusion method, whereas only 7% were released in 60 h when using the double emulsion method. A viability test on U-937 cells showed better survival rates with the particles prepared by the double emulsion technique. The results suggest that the location of the ODN in the polymeric matrix is affected by the encapsulation method. Particles containing CTAB appeared more toxic than the ones obtained by the double emulsion technique, however, these particles can still be used for antisense activity since high oligonucleotide loading can be achieved.

Stimulation of the B cell receptor, CD86 (B7-2), and the beta 2-adrenergic receptor intrinsically modulates the level of IgG1 and IgE produced per B cell.

Our findings using B cells from either wild-type, CD86-deficient, or beta 2-adrenergic receptor (beta2AR)-deficient mice suggest three mechanisms by which the level of IgG1 and IgE production can be increased on a per cell basis. Trinitrophenyl-specific B cells enriched from unimmunized mouse spleens were pre-exposed to Ag and/or the beta 2AR ligand terbutaline for 24 h before being activated by either a beta 2AR-negative Th2 cell clone or CD40 ligand/Sf9 cells and IL-4 in the presence or absence of an anti-CD86 Ab. Data suggest that the first mechanism involves a B cell receptor (BCR)-dependent up-regulation of CD86 expression that, when CD86 is stimulated, increases the amount of IgG1 and IgE produced in comparison to unstimulated cells. The second mechanism involves a BCR- and beta 2AR-dependent up-regulation of CD86 to a level higher than that induced by stimulation of either receptor alone that, when CD86 is stimulated, further increases the amount of IgG1 and IgE produced. The third mechanism is BCR-independent and involves a beta 2AR-dependent increase in the ability of a B cell to respond to IL-4. Flow cytometric and limiting dilution analyses suggest that the increase in IgG1 and IgE occurs independently from the isotype switching event. These findings suggest that the BCR, the beta 2AR, and CD86 are involved in regulating IL-4-dependent IgG1 and IgE production.

IL-4 induces serine phosphorylation of the STAT6 transactivation domain in B lymphocytes.

The binding of IL-4 to its receptor results in rapid tyrosine phosphorylation of STAT6 by IL-4R-associated Jak kinases. Phosphorylated STAT6 dimerizes and translocates to the nucleus where it acts as a transcription factor to regulate a number of important immune response-related genes in a variety of cell types. Studies of other STAT proteins have demonstrated a role for serine phosphorylation in addition to tyrosine phosphorylation in the regulation of STAT-mediated gene transcription. In this study, phosphoamino acid analysis and two-dimensional phosphopeptide mapping of STAT6 from mouse splenic B cells demonstrated that IL-4 induces phosphorylation of STAT6 on multiple serines. Expression and analysis of a mutant STAT6 protein in which tyrosine 641 (Y641) was replaced with phenylalanine demonstrated that Y641 is necessary for tyrosine phosphorylation of STAT6, but that tyrosine phosphorylation is not necessary for serine phosphorylation. Analysis of STAT6 deletion mutants localized the majority of serine phosphorylation sites to a region between residues 719 and 789, within the previously described transactivation domain. IL-4-stimulated serine phosphorylation of STAT6 was resistant to H7 and HA1004, inhibitors of many serine/threonine kinases including PKC. Serine phosphorylation was also resistant to Wortmannin and LY294002, demonstrating that the IRS/PI 3-kinase pathway is also not required. These data, coupled with previous studies showing that IL-4 does not activate MAPK pathways in lymphocytes, suggest that IL-4 may induce serine phosphorylation of STAT6 by a novel-signaling pathway.

Transcriptional repression of Stat6-dependent interleukin-4-induced genes by BCL-6: specific regulation of iepsilon transcription and immunoglobulin E switching.

The BCL-6 proto-oncogene encodes a POZ/zinc-finger transcription factor that is expressed in B cells and a subset of CD4(+) T cells within germinal centers. Recent evidence suggests that BCL-6 can act as a sequence-specific repressor of transcription, but the target genes for this activity have not yet been identified. The binding site for BCL-6 shares striking homology to the sites that are the target sequence for the interleukin-4 (IL-4)-induced Stat6 (signal transducers and activators of transcription) signaling molecule. Electrophoretic mobility shift assays demonstrate that BCL-6 can bind, with different affinities, to several DNA elements recognized by Stat6. Expression of BCL-6 can repress the IL-4-dependent induction of immunoglobulin (Ig) germ line epsilon transcripts, but does not repress the IL-4 induction of CD23 transcripts. Consistent with the role of BCL-6 in modulating transcription from the germ line epsilon promoter, BCL-6(-/-) mice display an increased ability to class switch to IgE in response to IL-4 in vitro. These animals also exhibit a multiorgan inflammatory disease characterized by the presence of a large number of IgE(+) B cells. The apparent dysregulation of IgE production is abolished in BCL-6(-/-) Stat6(-/-) mice, indicating that BCL-6 regulation of Ig class switching is dependent upon Stat6 signaling. Thus, BCL-6 can modulate the transcription of selective Stat6-dependent IL-4 responses, including IgE class switching in B cells.

In vitro antibacterial activity of thiamphenicol glycinate acetylcysteinate against respiratory pathogens.

After 30 years of therapeutic use, thiamphenicol glycinate acetylcysteinate (CAS 20192-91-0) is still widely employed in the treatment of upper and lower respiratory tract infections. This is due to its particular characteristic to exert at pulmonary level, either the antibacterial activity of thiamphenicol (CAS 15318-45-3) and the mucolytic activity of N-acetylcysteine (CAS 616-91-1). The aim of this study was to evaluate the present pattern of susceptibility of several clinical isolates to thiamphenicol and the interference of N-acetylcysteine on this parameter. The studies have been performed in vitro. Equimolar concentrations of N-acetylcysteine and even higher concentrations did not interfere with the antibacterial activity of thiamphenicol against Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae. The spectrum of activity of thiamphenicol was similar to that observed in the past and was superior to that of erythromycin and amoxicillin. The activity of thiamphenicol was greater than that of erythromycin against H. influenzae and streptococci and equivalent versus Branhamella catarrhalis. In comparison with amoxicillin the activity of thiamphenicol was higher against H. influenzae and B. catarrhalis and slightly lower against streptococci. The results demonstrate that thiamphenicol maintains its therapeutic value confirming the importance of thiamphenicol glycinate acetylcysteinate in the treatment of respiratory tract infections.

Regulation of the germline immunoglobulin Cgamma1 promoter by CD40 ligand and IL-4: dual role for tandem NF-kappaB binding sites.

Transcription of germline Ig constant region genes and associated switch regions is an early and essential step in heavy chain class switch recombination. Transcription of the germline Cgamma1 and C epsilon Ig genes is induced by IL-4 via STAT6 activation; CD40 signaling can independently induce transcription of these genes and act in synergy with IL-4 to increase expression. In the present study, we investigated the role of three tandem NF-kappaB sites (site 1, -95; site 2, -71; site 3, -53) in the regulation of the germline Cgamma1 Ig promoter by CD40 Ligand (CD40L) and IL-4 in the mouse B lymphoma cell line, BCL1-3B3. Germline gamma1 transcripts are induced by CD40L and by IL-4 in BCL1-3B3 and the combination of signals is synergistic, as in normal B cells. EMSA with crude nuclear extracts demonstrated that stimulation with CD40L results in the induction of NF-kappaB complexes that bind to each of the three NF-kappaB sites and are composed mainly of p50 and RelB, but also include c-Rel and p65. Surprisingly, site-specific mutagenesis of the NF-kappaB sites did not reduce CD40-responsiveness of germline gamma1 promoter-luciferase reporter constructs transiently transfected into BCL1-3B3. Mutation in any one NF-kappaB site, however, significantly reduced overall transcriptional activity of the promoter, both basal and induced, suggesting a role in basal promoter function. In addition, activation of the promoter by IL-4 was blocked by mutation of all three NF-kappaB sites and similarly reduced by mutation of site 1, suggesting that NF-kappaB-STAT6 interactions may be necessary for STAT6-mediated transactivation of the germline gamma1 promoter. The results suggest that the three NF-kappaB sites may serve as a focus for formation of a higher-order transcription complex including STAT6, NF-kappaB and components of the basal transcription apparatus.

Uptake of oligonucleotide-loaded nanoparticles in prostatic cancer cells and their intracellular localization.

The development of antisense biotechnology is dependent, in part, on creating improved methods for delivering oligonucleotides to cells. In this study, we investigated a colloidal system (nanoparticles (NP) of poly (D,L) lactic acid) that affects the intracellular delivery of oligonucleotides. We have examined the intracellular compartmentalization in DU145 cells of fluorescein labeled phosphorothioate oligonucleotides, both in the free state and when loaded into NP. Fluorescent oligonucleotides were incubated for 18 h with DU145 cells and the mean intracellular fluorescence was determined by flow cytometry. After the addition of monensin, an increase in signal intensity was observed, indicating that free oligonucleotides were resident in an acidic intracellular environment, whereas oligonucleotides from the NP did not reside in an acidic compartment. Free and NP loaded with oligonucleotides effluxed from DU145 cells from two intracellular compartments. This preliminary report indicates that colloidal carriers such as NP could prove to be useful in affecting intracellular trafficking of oligonucleotides in DU145 and in other cells.

Highly loaded nanoparticulate carrier using an hydrophobic antisense oligonucleotide complex.

Antisense oligonucleotides, and particularly those with phosphorothioate backbones, have emerged as potential gene specific therapeutic agents and are currently undergoing evaluation in clinical trials for a variety of diseases. In the area of HIV-1 therapeutics, targeting of oligonucleotides to infected cells, such as macrophages, would be highly desirable. The present study was designed to prepare and characterize oligonucleotide-loaded nanoparticles for this purpose. Due to their hydrophilic characteristics, oligonucleotides are difficult to entrap in polymeric particles. Here, the oligonucleotides were first complexed with cetyltrimethylammonium bromide. The oligonucleotide-loaded nanoparticles were prepared by the emulsification-diffusion method and subsequently purified. In comparison with previous studies, a high oligonucleotide-loading was achieved; 2.5, 5 and 10% oligonucleotide loading were assessed. If the initial oligonucleotide content was 4%, this method produced a final oligonucleotide loading of 1.9% with an entrapment efficiency of 47%. The integrity of the oligonucleotide and of the polymer, in the final freeze-dried product, was retained.

STAT6 is required for IL-4-induced germline Ig gene transcription and switch recombination.

Transcription of the germline C gamma1 and C epsilon Ig genes is believed to be a necessary prerequisite for isotype switching to IgG1 and IgE, respectively. IL-4 stimulation and ligation of CD40 can each independently induce low level germline gamma1 and epsilon transcription in murine B cells. Together these signals act synergistically to promote high level germline transcription and are normally required for T-dependent isotype switching to IgG1 and IgE. The STAT6 transcription factor has been suggested to play a critical role in IL-4-induced activation of germline C gamma1 and C epsilon genes. To directly assess the role of STAT6 in IL-4R- and CD40-mediated germline transcription and switching, we have analyzed these events in splenic B cells from STAT6-deficient mice. Our results demonstrate that IL-4 does not induce detectable levels of germline gamma1 or epsilon transcripts in STAT6-deficient B cells. Germline transcript expression induced by CD40 stimulation alone is unaffected, but synergism between CD40- and IL-4R-mediated signals is completely ablated. Switch recombination to S gamma1, as measured by digestion-circularization PCR, is dramatically reduced in STAT6-deficient B cells stimulated with CD40 ligand plus IL-4. Similarly, germline gamma1 transcript expression and switch recombination to S gamma1 are also impaired in STAT6-deficient B cells stimulated with IL-4, IL-5, and anti-IgD Abs conjugated to dextran, a model for T-independent type II responses. These results directly demonstrate a critical role for STAT6 in the IL-4-mediated activation of germline Ig gene transcription and switch recombination in nontransformed B cells.

CD40 expression by gingival fibroblasts: correlation of phenotype with function.

CD40, a member of the tumor necrosis factor-alpha receptor family, is constitutively expressed by cells of hematopoietic and non-hematopoietic origin, including fibroblasts. Signaling through this receptor molecule regulates inflammatory cytokine secretion by many cell types. Based on the recently described cytokine secretory heterogeneity of fibroblast cell subsets, we hypothesized that secretion of inflammatory cytokines by gingival fibroblast cultures may be dictated by the existence of differential proportions of cytokine-secreting subpopulations which express high levels of CD40. After examining a large number of gingival fibroblast (GF) cultures we find that the frequency of IL-6- and IL-8-secreting cells mirrors the frequency of cells expressing high levels of CD40 in these cultures. In addition, we demonstrate a direct functional relationship between CD40 expression and IL-6 or IL-8 secretion by showing that ligation of this molecule on GF, and CD40+ fibroblast subsets in particular, up-regulates secretion of these cytokines in vitro.