Intercellular adhesion molecule-1/LFA-1 ligation favors human Th1 development

Th cell polarization toward Th1 or Th2 cells is strongly driven by exogenous cytokines, in particular IL-12 or IL-4, if present during activation by Ag-presenting dendritic cells (DC). However, additional Th cell polarizing mechanisms are induced by the ligation of cell surface molecules on DC and naive Th cells. In the present study, the role of LFA-1/ICAM-1 ligation in human Th cell polarization was investigated. Triggering of LFA-1 on anti-CD3/CD28 stimulated naive Th cells with immobilized Fc-ICAM-1, in the absence of DC and exogenous cytokines, induced a marked shift toward Th1 cell development, accompanied by a dose-dependent decrease in GATA-3 expression and a dose-dependent increase in T-bet expression. Th1 polarization by LFA-1 ligation could be demonstrated only under low cytokine conditions, as it was largely overruled by IL-12 or IL-4. This IL-12-independent Th1-driving mechanism appears to be operated by certain subsets of effector DC. Maturation of DC by poly(I:C), a synthetic dsRNA, used as an in vitro model for viral infections, leads to the generation of Th1-driving effector DC (DC1), which express elevated levels of ICAM-1 but produce only low levels of IL-12p70. Blocking the ICAM-1/LFA-1 interaction in cocultures of these DC with naive Th cells attenuated their Th1-driving capacity. The molecular mechanism by which LFA-1 signaling supports Th1 differentiation is blocked by specific inhibitors of extracellular signal-regulated kinase phosphorylation. The present data indicate the existence of an IL-12-independent, extracellular signal-regulated kinase-mediated mechanism, through which high ICAM-1-expressing DC1 can drive Th1 polarization. This mechanism may be operational during viral infections.     

Pharmaco-toxicological study of Kageneckia oblonga, Rosaceae

The probable antipyretic, antiinflammatory, analgesic and antioxidant properties of Kageneckia oblonga, Rosaceae, were investigated and the major compounds of its active extracts were isolated. The study comprised the acute toxicity of the extracts of global methanol, hexane, dichloromethane and methanol. The cytotoxicity of global methanol extract was studied in three tumoral cell lines. All the extracts exhibited the pharmacological activities under study. Methanol and dichloromethane were the most toxic extracts. From the global methanol extract, isolations were performed of prunasin, 23,24- dihydro-cucurbitacin F, and a new cucurbitacin, 3beta-(beta-D-glucosyloxy)-16alpha,23alpha-epoxycucurbita-5,24-diene-11-one. The cytotoxicity of both cucurbitacins on human neutrophils at the assayed concentrations was not statistically significant. In-vitro assays showed that both cucurbitacins can be partly responsible for the analgesic, antipyretic, and anti-inflammatory activities. Evaluation was done of the cytotoxicity of global methanol extract, 23, 24-dihydrocucurbitacin F, aqueous extracts and prunasin against P-388 murine leukaemia, A-549 human lung carcinoma and HT-29 colon carcinoma. Since global methanol extract presented a strong cytotoxicity against P-388 murine leukaemia, A-549 human lung carcinoma, and HT-29 cell lines, it is highly probable that this extract contain one or more cytotoxic compounds that could be investigated for their potential use as an agent against cancer.     

Dendritic cells (DC) promote natural killer (NK) cell functions: dynamics of the human DC/NK cell cross talk

Dendritic cells (DC) were originally found critical in the setting of cognate immune responses. We first demonstrated that DC can also induce mouse NK cell activation and NK cell dependent-antitumor effects in mice. Here we analyzed the dynamics between DC and NK cells in human in vitro model systems. In the absence of LPS, DC do not trigger resting NK cells. Conversely, in the presence of LPS, resting bulk NK cells interacting with DC acquire CD25 and CD69 surface expression, produce high levels of IFN-gamma and lyse DAUDI cells. On activated IL-2 dependent NK cell lines, regardless of their differentiation stage, DC maintain or enhance NK cell proliferation and effector functions in the absence of exogenous cytokines. While IL-12, IL-15 and IL-18 are not critical, a direct cell-to-cell contact is mandatory for NK activation by DC and required for optimal proliferation. These data imply that DC also modulate human NK cell innate effector functions.     

Guanidinium and aminoimidazolinium derivatives of N-(4-piperidyl)propanamides as potential ligands for mu opioid and I2-imidazoline receptors: synthesis and pharmacological screening

Derivatives of N-(1-phenethyl-4-piperidyl)propanamides incorporating guanidinium and 2-aminoimidazolinium groups have been prepared by a synthetic approach involving first introduction of a spacer between the piperidine and the functional group by reductive amination of piperidinone. The formation of each of these functional groups was carried out using N-N'-di(tert-butoxycarbonyl)thiourea and 2-methylthioimidazolinium iodide, respectively. These structures have been designed to incorporate two pharmacologic goals into one entity. Radioligand binding assays have been used to study their affinity for opioid (mu, delta and kappa) and I2-imidazoline receptors. Two of them, 10 and 16, showed high affinity for mu opioid receptors and functionally they had moderate analgesic properties in the hot plate and writhing tests. The in vitro studies on guinea pig ileum (GPI) indicated that both compounds are mu opioid agonists. In what concerns I2-imidazoline receptor activity, these derivatives showed low affinity around 6 to 7 times less than idazoxan.     

Physical and functional interaction of the Arabidopsis K(+) channel AKT2 and phosphatase AtPP2CA

The AKT2 K(+) channel is endowed with unique functional properties, being the only weak inward rectifier characterized to date in Arabidopsis. The gene is expressed widely, mainly in the phloem but also at lower levels in leaf epiderm, mesophyll, and guard cells. The AKT2 mRNA level is upregulated by abscisic acid. By screening a two-hybrid cDNA library, we isolated a protein phosphatase 2C (AtPP2CA) involved in abscisic acid signaling as a putative partner of AKT2. We further confirmed the interaction by in vitro binding studies. The expression of AtPP2CA (beta-glucuronidase reporter gene) displayed a pattern largely overlapping that of AKT2 and was upregulated by abscisic acid. Coexpression of AtPP2CA with AKT2 in COS cells and Xenopus laevis oocytes was found to induce both an inhibition of the AKT2 current and an increase of the channel inward rectification. Site-directed mutagenesis and pharmacological analysis revealed that this functional interaction involves AtPP2CA phosphatase activity. Regulation of AKT2 activity by AtPP2CA in planta could allow the control of K(+) transport and membrane polarization during stress situations.     

TNFalpha and IL-1beta exert no direct vasoactivity in human isolated resistance arteries

The proinflammatory cytokines TNFalpha and IL-1beta are thought to play a role in the mechanism of septic vasodilatory shock. Most evidence indicate that the vascular effects these cytokines are mediated through increased expression of inducible nitric oxide synthase, whereas several animal studies report a more rapid vasorelaxant effect. We studied the direct vascular effects of TNFalpha and IL-1beta in human isolated resistance arteries (n=9) using a microvascular myograph.After precontraction with noradrenaline or a depolarising high potassium solution, TNFalpha and IL-1beta (up to 10(-8) M, alone or in combination) did not exert any relevant vasoactive effect. We conclude that, in contrast with some animal experiments, these cytokines exert no direct vasoactivity in human resistance arteries.     

Low-grade gliomas: clinical and pathobiological aspects

The optimal management of patients with low-grade gliomas remains a challenge for the treating physician. The natural history of the disease shows a large variety, and there is a substantial controversy about many of everyday treatment recommendations. However, new developments in clinical and basic research in neuro-oncology have occurred during the last years. In this review some of these new insights into clinical and biological aspects of low-grade gliomas are discussed, with focus on the translation of new knowledge from basic research into clinical practice. For example, molecular genetic profiling of tumour material has started to guide treatment recommendations and clinical management of some patients with oligodendrogliomas. Experimental studies of the different molecular pathways in tumour cells and in their normal counterparts involved in cell-cycle check-point control have elucidated some of the underlying mechanisms of resistance of gliomas to radiotherapy and chemotherapy. Finally, improved classification of the different subtypes of low-grade gliomas may be achieved in the near future by characterization of the genetic heterogeneity within the tumour and by identification of a putative stem cell as the origin of the tumour cells.     

Manufacturing DNA microarrays from unpurified PCR products

For the production of DNA microarrays from PCR products, purification of the the DNA fragments prior to spotting is a major expense in cost and time. Also, a considerable amount of material is lost during this process and contamination might occur. Here, a protocol is presented that permits the manufacture of microarrays from unpurified PCR products on aminated surfaces such as glass slides coated with the widely used poly(L-lysine) or aminosilane. The presence of primer molecules in the PCR sample does not increase the non-specific signal upon hybridisation. Overall, signal intensity on arrays made of unpurified PCR products is 94% of the intensity obtained with the respective purified molecules. This slight loss in signal, however, is offset by a reduced variation in the amount of DNA present at the individual spot positions across an array, apart from the considerable savings in time and cost. In addition, a larger number of arrays can be made from one batch of amplification products.     

Therapeutic efficacy of melanoma-reactive TIL injected in stage III melanoma patients

Adoptive therapy for cancer using tumor-infiltrating lymphocytes (TIL) has mainly been investigated in cancer patients with advanced stage disease. The limited clinical success has not been encouraging, although this might be explained by poor TIL specificity and/or high tumor burden. To re-evaluate the effectiveness of adoptive therapy, we analyzed the capacity of tumor-reactive TIL injection in preventing the further development of disease in stage III melanoma patients after complete tumor resection. A phase II/III randomized trial was performed on 88 melanoma patients, who received autologous TIL plus interleukin-2 (IL-2) or IL-2 only. The duration of relapse-free survival was analyzed, taking into account the immunological specificity of injected TIL and the number of metastatic lymph nodes removed before treatment. Kaplan-Meyer analysis revealed that the injection of tumor-reactive TIL was statistically correlated with prolonged relapse-free survival in patients with only one metastatic lymph node. Therefore, improved clinical outcome could be obtained after adoptive therapy by selecting appropriate groups of patients and monitoring the specificity of the injected TIL populations.