Ly49D engagement on T lymphocytes induces TCR-independent activation and CD8 effector functions that control tumor growth.

Recent data showing expression of activating NK receptors (NKR) by conventional T lymphocytes raise the question of their role in the triggering of TCR-independent responses that could be damaging for the host. Transgenic mice expressing the activating receptor Ly49D/DAP12 offer the opportunity to better understand the relevance of ITAM signaling in the biology of T cells. In vitro experiments showed that Ly49D engagement on T lymphocytes by a cognate MHC class I ligand expressed by Chinese hamster ovary (CHO) cells or by specific Ab triggered cellular activation of both CD4 and CD8 populations with modulation of activation markers and cytokine production. The forced expression of the ITAM signaling chain DAP12 is mandatory for Ly49D-transgenic T cell activation. In addition, Ly49D stimulation induced T lymphocyte proliferation, which was much stronger for CD8 T cells. Phenotypic analysis of anti-Ly49D-stimulated CD8 T cells and their ability to produce high levels of IFN-gamma and to kill target cells indicate that Ly49D ligation generates effector cytotoxic CD8 T cells. Ly49D engagement by itself also triggered cytotoxic activity of activated CD8 T cells. Adoptive transfer experiments confirmed that Ly49D-transgenic CD8 T cells are able to control growth of CHO tumor cells or RMA cells transfected with Hm1-C4, the Ly49D ligand normally expressed by CHO. In conclusion, Ly49D engagement on T cells leads to T cell activation and to a full range of TCR-independent effector functions of CD8 T cells.

Effect of collection and preprocessing methods on neutrophil elastase plasma concentrations.

OBJECTIVES: Elevated plasma levels of the elastase alpha 1-proteinase inhibitor complex (E-alpha 1 PI) have been proposed as a marker of bacterial infection and neutrophil activation. Liberation of elastase from neutrophils after collection of blood may cause falsely elevated results. Collection methods have not been validated for critically ill neonates and children. We evaluated the influence of preanalytical methods on E-alpha 1 PI results including the recommended collection into EDTA tubes. DESIGN AND METHODS: First, we compared varying acceleration speeds and centrifugation times. Centrifugation at 1550 g for 3 min resulted in reliable preparation of leukocyte free plasma. Second, we evaluated all collection tubes under consideration for absorption of E-alpha 1 PI. Finally, 12 sets of samples from healthy adults and 42 sets obtained from critically ill neonates and children were distributed into the various sampling tubes. Samples were centrifuged within 15 min of collection and analyzed with a new turbidimetric assay adapted to routine laboratory analyzers. RESULTS: One of the two tubes containing a plasma-cell separation gel absorbed 22.1% of the E-alpha 1 PI content. In the remaining tubes without absorption of E-alpha 1 PI no differences were observed for samples from healthy adult patients. However, in samples from critically ill neonates or children, significantly higher results were obtained for plain Li-heparin tubes (mean = 183 micrograms/L), EDTA tubes (mean = 93 micrograms/L), and citrate tubes (mean = 88.5 micrograms/L) than for the Li-hep tube with cell-plasma separation gel and no absorption of E-alpha 1 PI (mean = 62.4 micrograms/L, p < 0.01). CONCLUSION: Contrary to healthy adults, E-alpha 1 PI results in plasma samples from critically ill neonates and children depend on the type of collection tube.

Genetic diversity and ecological success of Staphylococcus aureus strains colonizing humans.

The genetic determinants and phenotypic traits which make a Staphylococcus aureus strain a successful colonizer are largely unknown. The genetic diversity and population structure of 133 S. aureus isolates from healthy, generally risk-free adult carriers were investigated using four different typing methods: multilocus sequence typing (MLST), amplified fragment length polymorphism analysis (AFLP), double-locus sequence typing (DLST), and spa typing were compared. Carriage isolates displayed great genetic diversity which could only be revealed fully by DLST. Results of AFLP and MLST were highly concordant in the delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. spa typing and DLST provided considerably less phylogenetic information. The resolution of spa typing was similar to that of AFLP and inferior to that of DLST. AFLP proved to be the most universal method, combining a phylogeny-building capacity similar to that of MLST with a much higher resolution. However, it had a lower reproducibility than sequencing-based MLST, DLST, and spa typing. We found two cases of methicillin-resistant S. aureus colonization, both of which were most likely associated with employment at a health service. Of 21 genotypic clusters detected, 2 were most prevalent: cluster 45 and cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carrier characteristics, such as age, sex, medical conditions, or antibiotic use. This may provide empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than those evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.

Programme latin de don d'organes: les intensivistes se mettent en réseau [Latin program for organ donation: the intensivists are networking]

The new Swiss federal law on organ and transplantation strengthens the responsibilities of the intensive care units. In Italian and French speaking parts of Switzerland, the Programme Latin pour le Don d'Organe (PLDO) has been launched to foster a wider collaboration between intensivists and donation coordinators. The PLDO aims at optimising knowledge and expertise in organ donation through improvements in identification, notification and management of organ donors and their next of kin. The PLDO dispenses education to all professionals involved. Such organisation should allow increasing the number of organs available, while improving healthcare professionals experience and next of kin emotion throughout the donation process.

Effect of antiviral therapy on circulating cytokeratin-18 fragments in patients with chronic hepatitis C.

Hepatocellular apoptosis plays a major role in the pathogenesis of chronic hepatitis C. It can be measured noninvasively by determining the circulating levels of cytokeratin-18 fragments. We hypothesized that the effect of antiviral therapy on this parameter will be different in patients with a sustained virological response, relapse (REL) and nonresponse (NR). We quantified cytokeratin-18 fragments in plasma of patients participating in the Swiss Hepatitis C cohort, who received antiviral therapy without stopping because of sides effects. A total of 315 patients were included, 183 with a sustained response, 64 with NR and 68 who relapsed. Mean levels ±SD of circulating cytokeratin-18 fragments before therapy were 174 ± 172 U/L for responsders, 188 ± 145 for nonresponders and 269 ± 158 U/L for patients who relapsed. The values were significantly higher in the REL group (ANOVA P < 0.006). A sustained response was associated with a significant improvement of the plasma levels (94 ± 92 U/L, paired test P < 0.000001), whereas there was no improvement in the nonresponder group (183 ± 158 U/L) and in the relapser group (158 ± 148 U/L). There was a weak correlation between alanine aminotransferase (ALT) and cytokeratin-18 fragment levels (r² = 0.35, P < 0.000001) before therapy but not after therapy and none with hepatitis C virus (HCV) viremia. Successful antiviral therapy results in a significant decrease in circulating levels of cytokeratin-18 fragments arguing for a reduction in hepatocellular apoptosis after clearance of the HCV. Baseline cytokeratin-18 fragment levels are higher in relapsers. Correlations with ALT are weak, suggesting that these two tests measure different but related processes.

Protein kinase C-zeta mediates retinal degeneration in response to TNF.

Tumor necrosis factor-alpha (TNF) has been implicated in retinal ganglion cells (RGC) degeneration in glaucoma. Atypical protein kinase C (PKC) zeta is involved in cell protection against various stresses. The aim of this study was to investigate the potential proapoptotic effects of intravitreal injections of TNF with or without PKCzeta specific inhibitor on the rat retina. TNF was injected in the vitreous of rat eyes alone or in combination with specific PKCzeta inhibitor. PKCzeta and NF-kappaB were studied by immunohistochemistry and western-blotting analysis on retina, and apoptosis quantified by the TUNEL assay. While low basal PKCzeta was observed in the control eyes, TNF induced intense expression of PKCzeta mostly in bipolar cells processes. PKCzeta staining became nuclear when TNF was coinjected with PKCzeta inhibitor. TNF alone did not induce apoptosis in the retina. Coinjection of the PKCzeta-specific inhibitor and TNF, however, induced apoptosis in the inner nuclear and ganglion cell layers. The PKCzeta-specific inhibitor unmasks retinal cells to TNF cytotoxicity showing a link between the proapoptotic effects of TNF and the antiapoptotic PKCzeta signaling pathway.

Fracture ostéoporotique chez les femmes ménopausées: l'alendronate réduit le risque. [Osteoporotic fracture in menopausal women: alendronate reduces the risk]

Cette rubrique présente les résultats d'une revue systématique publiée par la Collaboration Cochrane dans la Cochrane Library au premier trimestre 2008 (www.cochrane.org/). Volontairement limité à un champ de recherche circonscrit, cet article reflète l'état actuel des connaissances de ce domaine. Il ne s'agit donc pas de recommandations pour guider la prise en charge d'une problématique clinique considérée dans sa globalité (guidelines).

Acúmulo de ácido oxálico e cristais de cálcio em ectomicorrizas de eucalipto.: I- produção de ácido oxálico e concentração de nutrientes em raízes laterais finas colonizadas por fungos ectomicorrízicos

O eucalipto apresenta ótimo crescimento em solo com baixa fertilidade, mas pouco se sabe sobre a participação das ectomicorrizas e de ácidos orgânicos na aquisição e no acúmulo de nutrientes pela planta em campo. A produção de ácido oxálico e sua relação com as concentrações de P, Ca, Mg e K foram avaliadas em ectomicorrizas e raízes laterais finas de híbrido de Eucalyptus grandis x Eucalyptus urophylla, de 2,5 anos de idade, na região de Viçosa, MG. A área de estudo apresenta topografia típica em meia laranja, de vertente côncavo-convexa. Foram também avaliadas as concentrações desse composto no solo rizosférico, não rizosférico e ectomicorrizosférico. As maiores percentagens de colonização micorrízica foram observadas na área de encosta, onde havia limitada disponibilidade de nutrientes e alta saturação de Al. As concentrações de ácido oxálico + oxalato corresponderam, em mg kg-1: folhas, 324,6; ectomicorrizas, 208,3; raízes laterais finas não colonizadas, 183,1. Já no solo, as concentrações foram maiores no solo ectomicorrizosférico, com 183,7 mg kg-1, seguido pelo solo rizosférico, com 134,3 mg kg-1, e pelo solo não rizosférico, com 76,0 mg kg-1. As maiores concentrações de ácido oxálico e P (p < 0,05) nas ectomicorrizas da área do topo, 117,3 mg kg-1 e 6,3 g kg-1, respectivamente, sugerem que as populações de fungos ectomicorrízicos nesta área têm papel importante na solubilização e disponibilização de nutrientes para o hospedeiro. Não foram observadas correlações positivas significativas entre a produção de ácido oxálico e as concentrações de Ca nas raízes laterais finas e nas ectomicorrizas de eucalipto.

Development of a high throughput PCR to detect Coxiella burnetii and its application in a diagnostic laboratory over a 7-year period.

Q fever is a worldwide zoonotic infectious disease due to Coxiella burnetii. The clinical presentation may be acute (pneumonia and/or hepatitis) or chronic (most commonly endocarditis). Diagnosis mainly relies on serology and PCR. We therefore developed a quantitative real-time PCR. We first tested blindly its performance on various clinical samples and then, when thoroughly validated, we applied it during a 7-year period for the diagnosis of both acute and persistent C. burnetii infection. Analytical sensitivity (< 10 copies/PCR) was excellent. When tested blindly on 183 samples, the specificity of the PCR was 100% (142/142) and the sensitivity was 71% (29/41). The sensitivity was 88% (7/8) on valvular samples, 69% (20/29) on blood samples and 50% (2/4) on urine samples. This new quantitative PCR was then successfully applied for the diagnosis of acute Q fever and endovascular infection due to C. burnetii, allowing the diagnosis of Q fever in six patients over a 7-year period. During a local small cluster of cases, the PCR was also applied to blood from 1355 blood donors; all were negative confirming the high specificity of this test. In conclusion, we developed a highly specific method with excellent sensitivity, which may be used on sera for the diagnosis of acute Q fever and on various samples such as sera, valvular samples, aortic specimens, bone and liver, for the diagnosis of persistent C. burnetii infection.

Estoques de carbono no solo e na biomassa em plantações de eucalipto

O eucalipto é a principal espécie florestal plantada no Brasil. Todavia, são escassas informações acerca do C estocado no solo e na biomassa desses plantios. Este trabalho teve como objetivos avaliar o estoque de C no solo (ECS) em plantações de eucalipto e determinar quais características edafoclimáticas determinam esse estoque. O estudo foi conduzido em eucalipto cultivado na região centro-leste do Estado de Minas Gerais, abrangendo cinco regiões: Cocais (CO), Rio Doce (RD), Sabinópolis (SA), Santa Bárbara (SB) e Virginópolis (VI). Foi calculado o estoque de C no solo até 100 cm de profundidade em plantações de eucalipto em áreas com predomínio de seis classes de solo: Cambissolo Háplico (CX), Latossolo Amarelo (LA), Latossolo Vermelho (LV), Latossolo Vermelho-Amarelo (LVA), Neossolo Flúvico (RU) e Plintossolo Pétrico (FF). Os estoques de C no solo variaram entre regiões e classes de solo. O maior ECS ocorreu no LV, com 183,07 t ha-1 de C, seguido pelas classes de CX, LVA, LA, FF e RU, com 135,65, 130,95, 121,58, 112,01 e 95,08 t ha-1 de C, respectivamente. Em relação ao estoque médio de C no solo por região, considerando todas as classes de solo, o maior ECS foi de 141,22 t ha-1; determinado na região de VI, na profundidade até 100 cm de profundidade, seguida pelas regiões SA, CO, SB e RD, com 135,54, 127,26, 112,89 e 80,79 t ha-1 de C, respectivamente. Ao se considerar o estoque de C total no sistema solo-planta, aos 84 meses de idade, a região de SA foi a que apresentou maior estoque, com 251,61 t ha-1; e a região de RD, o menor estoque, com 186,84 t ha-1 de C. O ECS pode ser estimado por equações compostas por características edofoclimáticas, sendo a variação em ECS explicada por características como teor de argila e de Al3+; pela altitude e pelo déficit hídrico da região.