Toll-like receptor 4 gene polymorphism influences dendritic cell in vitro function and clinical outcomes in vaccinated melanoma patients

Toll-like receptor 4 (TLR4) is expressed on dendritic cells (DCs), sensing environmental danger molecules that induce their activation and maturation. Recently, we reported a method for the production of therapeutic DCs against melanoma, called tumor antigen-presenting cells (TAPCells), using a heat-shocked allogeneic melanoma cell lysate (TRIMEL) as an activation factor and antigen provider. Since TRIMEL contains endogenous TLR4 ligands, we evaluated the role of TLR4 in TAPCells differentiation by antibody neutralization and the association of a Tlr4 polymorphism (896A/G) (Asp299Gly), determined by PCR–RFLP, with the in vitro activation capacity and the clinical outcome of TAPCells-vaccinated patients. Antibody blocking of monocyte TLR4 inhibited surface expression, determined by flow cytometry, of the major histocompatibility complex class I, CCR7, CD80, CD83 and CD86 on TAPCells, reduced interleukin (IL)-6 and tumor necrosis factor -α gene expression evaluated by qRT-PCR, and also inhibited the TAPCells-mediated interferon-γ (IFN-γ) secretion of melanoma-specific CD8⁺ T cells determined by ELISpot (p?<?0.01). Moreover, CD8⁺ T-cell activation capacity was significantly reduced in TAPCells bearing the TLR4 Asp299Gly receptor (p?<?0.05). Finally, TAPCells-vaccinated stage-IV melanoma patients bearing the Tlr4 896G allele showed a shortened post-therapy median survival rate compared with those carrying the Tlr4 896A allele (p?<?0.05; log-rank test). Our results indicate that TLR4 is a key receptor for the tumor lysate-mediated in vitro generation of clinically efficient antigen-presenting cells. Further analysis of patients included in different vaccine protocols is necessary for definitively establishing a role for TLR4 polymorphism in clinical responses.     

Suppression of estrus in cats with melatonin implants

The objective of this study was to assess the efficacy of a subcutaneous melatonin implant to suppress estrus in queens (felis catus). The hypothesis was that this implant would temporarily and reversibly suppress estrus in queens without producing any clinically detectable side effects. Fourteen adult queens were maintained in cages under artificial illumination (14 h light:10 h dark) for 45 d and then randomly assigned to one of two treatments. At interestrus, queens received a single subcutaneous melatonin implant (18 mg; Melovine [CEVA Sante Animal, Libourne, France]; MEL: n = 9), or a single subcutaneous placebo implant without melatonin (0 mg; PLA; n = 5). At the next estrus, all queens received a second MEL (n = 9) or PLA (n = 5) implant. Blood samples were taken when queens displayed estrous signs and during interestrus to measure estradiol (E₂) and progesterone (P₄), respectively, by radioimmunoassay. There were no significant differences in duration of the interestrus interval in PLA cats, regardless of whether the implants were placed during interestrus or estrus (6.0 ± 9.7 d vs. 6.0 ± 9.7 d, respectively; least square means [LSM] ± SEM). However, when MEL implants were placed during interestrus, the duration of interestrus was approximately twice as long as that occurring when MEL implants were placed during estrus (113.3 ± 6.1 d vs. 61.1 ± 6.8 d, respectively; P < 0.01). Serum E₂ and P₄ concentrations were similar in queens with PLA and MEL implants and in queens that received implants in estrus and interestrus. In conclusion, a subcutaneous MEL implant effectively and reversibly suppressed estrus in queens for approximately 2 to 4 mo with no clinically detectable side effects.     

Genetic structure reveals management units for the yellow cardinal (<Emphasis Type="Italic">Gubernatrix cristata</Emphasis>), endangered by habitat loss and illegal trapping

The yellow cardinal, Gubernatrix cristata, is an endangered passerine from southern South America. Populations are declining due to the loss of their natural habitat, which has caused a fragmented distribution, and the continuous extraction of individuals from the wild, mainly males, to sell them as cage birds. In this study, we assess the genetic variability of remaining yellow cardinal’s populations and determine whether these populations represent independent management units. We found that the degree of geographic isolation of the remaining populations parallels the genetic differentiation of these populations for both mitochondrial and nuclear markers, and supports the delimitation of four management units for the yellow cardinal (three in Argentina and one in Uruguay). Assignment tests showed that geographic genetic differentiation can be used to assign seized individuals from illegal pet trade to their original populations and thus manage their release.     

Nitrogen uptake by crops,soil distribution and recovery of urea-N in a sorghum—wheat rotation in different soils under Mediterranean conditions

The N uptake by crops, soil distribution and recovery of 15N labelled urea-N (100 kg N ha-1) were investigated in a sorghum-wheat rotation in two silty clay soils (Foggia and Rieti Casabianca) and one silt loam soil (Rieti Piedifiume) under different mediterranean conditions. Non-exchangeable labelled NH4-N represented an important pool at both Rieti sites with higher values (p<0.05) under sorghum (14.0 and 24.6% of the urea N in the 0-20 cm layer at the end of the cropping season) than wheat whereas it was much less important in the Foggia soil (10.0% in the surface soil under sorghum). This is probably related to the clay minerals composition of the three soils; because vermiculite was present in both Rieti sites but not in the Foggia soil. At harvest from 4.4 to 5.3% of the urea N initially applied was present as microbial biomass N in the surface soil layer with no generally significant differences due to location and type of crops. Both sorghum and wheat N yields were higher in the driest site (Foggia) probably due to better light conditions, higher temperatures and irrigation during summer of the sorghum cropping period. The recovery of plant fertilizer N (about 21% for sorghum and 27% for wheat) and the percentage of N in the plant derived from the fertilizer (NDFF) were the lowest at Rieti-Casabianca probably as the result of the protection of immobilized fertilizer N against microbial mineralization by the swelling clays. The fertilizer N unaccounted for was nil or very low (10.8% at Rieti-Casabianca under wheat and 11.8 and 4.9% at Rieti-Piedifiume under sorghum and wheat, respectively). Urea-N losses occurred when Rieti Piedifiume and Rieti Casabianca soils were kept bare. In this case the urea N unaccounted for ranged from 12 to 56% of the urea N with higher losses in Rieti-Piedifiume than in Rieti-Casabianca. The higher recoveries in the latter soil were probably confirmed by the stabilizing effect of clays on the immobilized urea N. This revised version was published online in July 2006 with corrections to the Cover Date.     

Dengue Virus 1 in Buenos Aires from 1999 to 2010: Towards Local Spread

Dengue virus (DENV) is a public health problem representing the most important arthropod-borne viral disease in humans. In Argentina, Northern provinces have reported autochthonous cases since 1997, though these outbreaks have originated in bordering countries, where co-circulation of more than one serotype has been reported. In the last decade, imported dengue cases have been reported in Buenos Aires, the urban area of Argentina with the highest population density. In 2009, a dengue outbreak affected Buenos Aires and, for the first time, local transmission was detected. All cases of this outbreak were caused by DENV-1. In this report, we present the full-length sequences of 27 DENV-1 isolates, corresponding to imported cases of 1999–2000, as well as local and imported cases of the 2009 and 2010 outbreaks. We analyzed their phylogenetic and phylodynamic relationships and their global and local spread. Additionally, we characterized their genomic and phenotypic features. All cases belonged to DENV-1 genotype V. The most recent ancestor for this genotype was dated ∼1934, whereas that for the 2009 outbreak was dated ∼2007. The mean rates of nucleotide substitution were 4.98E-4 and 8.53E-4 subs./site/yr, respectively. We inferred an introduction from Paraguay in 1999–2000 and mainly from Venezuela during 2009–2010. Overall, the number of synonymous substitutions per synonymous site significantly exceeded the number of non-synonymous substitutions per site and 12 positively selected sites were detected. These analyses could contribute to a better understanding regarding spread and evolution of this pathogen in the Southern Cone of South America.     

Isolation and functional characterization of cold-regulated promoters, by digitally identifying peach fruit cold-induced genes from a large EST dataset

Background  

Cold acclimation is the process by which plants adapt to the low, non freezing temperatures that naturally occur during late autumn or early winter. This process enables the plants to resist the freezing temperatures of winter. Temperatures similar to those associated with cold acclimation are also used by the fruit industry to delay fruit ripening in peaches. However, peaches that are subjected to long periods of cold storage may develop chilling injury symptoms (woolliness and internal breakdown). In order to better understand the relationship between cold acclimation and chilling injury in peaches, we isolated and functionally characterized cold-regulated promoters from cold-inducible genes identified by digitally analyzing a large EST dataset.     

Effect of storage media and storage time on survival of spermatozoa recovered from canine and feline epididymides

The aim of this study was evaluate the survival ability of canine and feline spermatozoa maintained within epididymides stored at 4 degrees C for 24, 48 or 72 h in sterile isotonic saline solution (SAL) or a Tris-egg yolk (TEY) storage medium. Fifteen domestic dogs and 15 cats were neutered and their testes were placed in TEY or SAL and stored at 4 degrees C for either 24, 48 or 72 h. Sperm samples were obtained by cutting the cauda epididymides into a Tris extender and were evaluated for motility, velocity, viability, plasma membrane integrity, and acrosome morphology. In dogs, there were no significant differences between storage media for motility, plasma membrane integrity, viability and velocity. However, dog sperm stored in TEY had better acrosome morphology compared to sperm stored in SAL (P < 0.05). Dog sperm recovered at 72 h had a reduction in all parameters studied compared to those recovered at 24 h (P < 0.05). In cats, sperm recovered from epididymides stored in TEY had higher motility, plasma membrane integrity and velocity at all times compared to those stored in SAL (P < 0.05). Cat sperm recovered at 72 h had reduced motility, acrosome morphology, viability and velocity compared to those recovered at 24 h (P < 0.05). The addition of TEY to canine epididymal sperm, thus, had a better protective effect than SAL only on acrosome morphology. In cats, in contrast, TEY had a better protective effect than SAL on all epididymal sperm parameters studied. In both species, sperm recovered at 72 h had a significant reduction in all parameters studied compared to those recovered at 24 h.     

The immunological response and post-treatment survival of DC-vaccinated melanoma patients are associated with increased Th1/Th17 and reduced Th3 cytokine responses

Introduction

Immunization with autologous dendritic cells (DCs) loaded with a heat shock-conditioned allogeneic melanoma cell lysate caused lysate-specific delayed type hypersensitivity (DTH) reactions in a number of patients. These responses correlated with a threefold prolonged long-term survival of DTH⁺ with respect to DTH^? unresponsive patients. Herein, we investigated whether the immunological reactions associated with prolonged survival were related to dissimilar cellular and cytokine responses in blood.

Materials and methods

Healthy donors and melanoma patient’s lymphocytes obtained from blood before and after vaccinations and from DTH biopsies were analyzed for T cell population distribution and cytokine release.

Results/discussion

Peripheral blood lymphocytes from melanoma patients have an increased proportion of Th3 (CD4⁺ TGF-β⁺) regulatory T lymphocytes compared with healthy donors. Notably, DTH⁺ patients showed a threefold reduction of Th3 cells compared with DTH^? patients after DCs vaccine treatment. Furthermore, DCs vaccination resulted in a threefold augment of the proportion of IFN-γ releasing Th1 cells and in a twofold increase of the IL-17-producing Th17 population in DTH⁺ with respect to DTH^? patients. Increased Th1 and Th17 cell populations in both blood and DTH-derived tissues suggest that these profiles may be related to a more effective anti-melanoma response.

Conclusions

Our results indicate that increased proinflammatory cytokine profiles are related to detectable immunological responses in vivo (DTH) and to prolonged patient survival. Our study contributes to the understanding of immunological responses produced by DCs vaccines and to the identification of follow-up markers for patient outcome that may allow a closer individual monitoring of patients.     

Citrus compost and its water extract for cultivation of melon plants in greenhouse nurseries. Evaluation of nutriactive and biocontrol effects

Two different types of citrus composts, and their water extracts, were tested with regard to their utilisations as partial substitutes for peat in growing media for melon seedlings in greenhouse nurseries. Both compost showed higher plant growth than peat. Compost composed by citrus waste and green residue (C2) showed greater plant growth than compost obtained from the same organic matrices mentioned above further the addition of sludge obtained from citrus industry (C1). Compost C2 showed a greater auxinic effect than C1 and it was the only one that showed cytokinic effect. Both composts also demonstrated a biocontrol effect against Fusarium oxysporum for melon plants: the effects were also higher in C2 than in C1. Higher number of isolated fungi was active against F. oxysporum in compost C2, than compost C1. No different bacterial biocontrol efficacy was observed between both composts. The water extracts of both composts gave lower plant yields than their solid matrices, their relative effects being similar to those of the solid composts (C2 extract gave higher plant yields than the extract from C1). The biocontrol effects of compost water extracts followed the same trend.