Protein kinase C-theta is required for development of experimental cerebral malaria

Cerebral malaria is the most severe neurologic complication in children and young adults infected with Plasmodium falciparum. T-cell activation is required for development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (CM). To characterize the T-cell activation pathway involved, the role of protein kinase C-theta (PKC-θ) in experimental CM development was examined. PKC-θ-deficient mice are resistant to CM development. In the absence of PKC-θ, no neurologic sign of CM developed after blood stage PbA infection. Resistance of PKC-θ-deficient mice correlated with unaltered cerebral microcirculation and absence of ischemia, as documented by magnetic resonance imaging and magnetic resonance angiography, whereas wild-type mice developed distinct microvascular pathology. Recruitment and activation of CD8(+) T cells, and ICAM-1 and CD69 expression were reduced in the brain of resistant mice; however, the pulmonary inflammation and edema associated with PbA infection were still present in the absence of functional PKC-θ. Resistant PKC-θ-deficient mice developed high parasitemia, and died at 3 weeks with severe anemia. Therefore, PKC-θ signaling is crucial for recruitment of CD8(+) T cells and development of brain microvascular pathology resulting in fatal experimental CM, and may represent a novel therapeutic target of CM.     

IL-22 is produced by γC-independent CD25+ CCR6+ innate murine spleen cells upon inflammatory stimuli and contributes to LPS-induced lethality

IL-22 is a Th17 cytokine that plays a key role in immune responses against extracellular bacteria. In mucosal lymphoid tissues, IL-22 production is mainly due to an IL-23-responsive NK-like cell subset that shares some markers with lymphoid tissue inducer (LTi) cells. Here, we identified a new spleen cell population responsible for IL-22 production upon either in vitro stimulation by anti-CD3 antibodies or in vivo stimulation by lipopolysaccharide (LPS) via IL-2- and an IL-23-dependent mechanisms, respectively. These cells represent 1% of spleen cells from recombination activating gene (Rag2)-deficient mice, and correspond to a discrete innate lymphoid cell population expressing CD25, CCR6 and IL-7R. This population comprises 60-70% CD4(+) cells, which produce IL-22, and are still present in common γ chain-deficient mice; the CD4(-) subset coexpresses IL-22 and IL-17, and is common γ chain-dependent. The importance of IL-22 production for the LPS-triggered response is highlighted by the fact that IL-22-deficient mice are more resistant to LPS-induced mortality.     

Protein kinase LegK2 is a type IV secretion system effector involved in endoplasmic reticulum recruitment and intracellular replication of Legionella pneumophila

Legionella pneumophila is the etiological agent of Legionnaires' disease. Crucial to the pathogenesis of this intracellular pathogen is its ability to subvert host cell defenses, permitting intracellular replication in specialized vacuoles within host cells. The Dot/Icm type IV secretion system (T4SS), which translocates a large number of bacterial effectors into host cell, is absolutely required for rerouting the Legionella phagosome. Many Legionella effectors display distinctive eukaryotic domains, among which are protein kinase domains. In silico analysis and in vitro phosphorylation assays identified five functional protein kinases, LegK1 to LegK5, encoded by the epidemic L. pneumophila Lens strain. Except for LegK5, the Legionella protein kinases are all T4SS effectors. LegK2 plays a key role in bacterial virulence, as demonstrated by gene inactivation. The legK2 mutant containing vacuoles displays less-efficient recruitment of endoplasmic reticulum markers, which results in delayed intracellular replication. Considering that a kinase-dead substitution mutant of legK2 exhibits the same virulence defects, we highlight here a new molecular mechanism, namely, protein phosphorylation, developed by L. pneumophila to establish a replicative niche and evade host cell defenses.     

Expression of Integrin α6β1 Enhances Tumorigenesis in Glioma Cells

The integrin α6β1 and its main ligand laminin-111 are overexpressed in glioblastoma, as compared with normal brain tissue, suggesting they may be involved in glioblastoma malignancy. To address this question, we stably expressed the α6 integrin subunit in the U87 cell line via retroviral-mediated gene transfer. We show that cell surface expression of the α6β1 integrin led to dramatic changes in tumor U87 cell behavior, both in vitro and in vivo. Nude mice receiving either subcutaneous or intracerebral inoculation of α6β1-expressing cells developed substantially more voluminous tumors than mice injected with control cells. The difference in tumor growth was associated with a marked increase in vascularization in response to α6β1 integrin expression and may also be related to changes in the balance between cell proliferation and survival. Indeed, expression of α6β1 enhanced proliferation and decreased apoptosis of U87 cells both in the tumor and in vitro. Additionally, we demonstrate that α6β1 is implicated in glioblastoma cell migration and invasion and that laminin-111 might mediate dissemination of α6β1-positive cells in vivo. Our results highlight for the first time the considerable role of the integrin α6β1 in glioma progression.Malignant brain tumors have an increasing incidence in both children and adults. In adults, the most common type of primary brain tumor, malignant glioma, is considered as one of the deadliest of human cancers. Despite recent advances in both diagnostic modalities and therapeutic strategies, the 5-year survival rate of less than 3% in patients with glioblastoma is among the lowest for all cancers.¹ Patients with the most malignant histopathological subtype, glioblastoma, carry the worst prognosis, with median survival rate of less than 1 year, despite aggressive surgery associated with adjuvant radiotherapy and chemotherapy.¹ Glioblastoma are characterized by rapidly dividing cells, high degree of vascularity, invasion into normal brain tissue, and an intense resistance to death-inducing stimuli.^2,3 Since integrins, the major family of extracellular matrix (ECM) receptors, are involved in these events, they are one of the most promising molecules to consider for a targeted therapy.Integrins are cell surface transmembrane αβ heterodimers that recognize specific ECM ligands. The combination of α and β subunits, leading to the formation of at least 24 receptors, determines the ligand specificity.⁴ Glioblastoma commonly displays enhanced expression of several integrins along with their ECM ligands: αvβ3 and αvβ5 (tenascin and vitronectin receptors), α5β1 (fibronectin receptor), α2β1 (collagens receptor), and α3β1, α6β4, and α6β1 (laminins receptors).⁵ Numerous studies have focused on the αv integrin family. The integrins αvβ3 and αvβ5 are markers of glioblastoma malignancy⁶ and influence a variety of processes in glioblastoma progression in vivo, including proliferation, apoptosis, and angiogenesis.⁷ Furthermore, cilengitide, an αvβ3 and αvβ5 integrins antagonist, extends mouse survival by delaying the tumor growth^8,9 and is nowadays in clinical trial for recurrent malignant glioma. Two other integrins, α5β1 and α3β1, have been shown to be implicated in glioma cell adhesion and migration in vitro.^10,11 In addition, the use of α5β1 antagonists reduces glioma cell proliferation in vitro,¹⁰ while α3β1 antagonists inhibited glioma invasion in vivo.¹¹The α6 integrin subunit associates with β1 or β4 subunits to form functional heterodimers that selectively bind laminins. The α6β4 integrin is essential for the organization and maintenance of epithelial hemidesmosomes that link the intermediate filaments with the extracellular matrix.¹² The major ligand of α6β4 is the laminin-332, while α6β1 is a well-characterized laminin-111 receptor. Overexpression of α6β1 integrin has been associated with the progression of many epithelial tumors. In particular, induction of α6β1 expression is an early event in hepatocellular carcinogenesis.^13,14 In the same way, during prostate cancer progression α6β1 is continually expressed and found in micrometastases.¹⁵ Expression of α6β1 integrin has also been linked to metastatic potential of melanoma cells,¹⁶ and has been involved in the survival and metastatic potential of human breast carcinoma cells.^17,18 Moreover, in a recent study using the α6-blocking antibody GoH3, Lee et al¹⁹ inhibited angiogenesis and breast carcinoma growth in vivo.Several studies concerning gliomas and the α6β1 ligand laminin-111 have been reported in the literature. Using immunohistochemistry studies, Gingras et al²⁰ showed that α6 integrin was strongly expressed in glioblastoma tissue, whereas it was weakly expressed in normal brain. Previtali et al²¹ confirmed that the expression of α6 was increased in glioblastoma and in other central nervous system tumors, such as meningioma, astrocytoma, and neuroblastoma, when compared with the autologous normal tissue counterpart. In glioblastoma biopsies, laminin-111 is highly expressed on tumor blood vessels, but also within the brain tumor as punctuate deposits and at the tumor invasion front.²² In vitro, glioma cells can both secrete laminin-111 and induce its expression in normal brain tissue.^22,23,24 Moreover, laminin-111 is one of the most permissive substrates for adhesion and migration of glioma cells in vitro.^25,26,27 Additionally, over laminin-111, migrating glioma cells are protected from apoptosis.²⁸ For all these reasons, we hypothesized that laminin-111 and its main receptor α6β1 may contribute to glioblastoma progression.In the present study we investigated the role of integrin α6β1 in glioblastoma malignancy by using U87, a well-characterized glioblastoma cell line. We report that stable expression of α6β1 in this α6-negative cell line leads to enhanced tumor progression and tumor growth in vivo. We demonstrate that α6β1 is pro-angiogenic and acts on the balance between proliferation and apoptosis. Additionally, we show that α6β1 is involved in glioblastoma cell migration and invasion. Our results highlight for the first time the considerable role of integrin α6β1 in the malignant phenotype of glioblastoma cells and demonstrate that the α6β1-expressing cell is an appropriate model for the study of glioblastoma progression.     

Shaping home care in Europe: The contribution of the Aged in Home Care project

Objectives

During the 1990s, use of home care sector has increased substantially in Europe. However, research on home care continues to be underreported. This article summarizes the findings from the “Aged in Home Care” (ADHOC) study – carried out from 2001 to 2004 in Europe – and women's situation in European Home Care.

Methods

The review is based on 4 book chapters as well as on 23 articles listed in PubMed and published from August 2004 to October 2008. ADHOC used a standardized data set collected with the Resident Assessment Instrument for Home Care (RAI-HC 2.0); this instrument was used to assess 4010 home care clients at 11 European sites. The included articles analyzed the sociodemographic and clinical characteristics, basic physical needs, provision of selected preventive measures, and medication data from the ADHOC sample. In addition home service provision, quality indicators, and selected outcomes of home care intervention during the course of 1 year were assessed.

Results

The mean subject age was 82.3 years; women were on average 2 years older than men and more frequently lived alone, 74% were women. Women suffered more frequently from pain, depression, and extreme obesity. There were marked regional differences in both the functional status of the clients and the characteristics and use of home care services.

Conclusions

The implementation of a common assessment instrument for HC clients may help contribute the necessary wealth of data for (re)shaping home care in Europe. Policy makers and service providers may learn about best practices in the European context.     

Oxidized low-density lipoproteins in cord blood from neonates with intra-uterine growth restriction

Objective

We verified whether oxidative stress indices (oxidized low-density lipoproteins and malondialdehyde) and inflammatory biomarkers (circulating C-reactive protein, interleukin-6, tumour necrosis factor-α, serum amyloid A and soluble intercellular vascular cell adhesion molecule) are increased in the umbilical vein of placental insufficiency induced intra-uterine growth restricted neonates.

Study design

The prospective cohort study, involving 3 tertiary care centers, consists of 200 consecutively recruited pregnant women carrying twins. We chose the twin pregnancy model because both fetuses share the same maternal environment, thereby avoiding potential confounding factors when comparing oxidative stress and inflammation biomarkers. We analysed only twin pairs with one with intra-uterine growth restriction (N = 38) defined as fetal growth < 10th percentile with abnormal Doppler of the umbilical artery. Blood samples were taken at birth from the umbilical vein. Intra-pair comparisons on the biomarkers were performed using the Student paired t-test.

Results

We observed increased cord blood levels of oxidized low-density lipoproteins, (2.394 ± .412 vs 1.296 ± .204, p = .003) but not of malondialdehyde in growth restricted neonates when compared to their normal counterparts. Although indices of inflammation tended to be increased in cord blood from growth restricted newborns, the difference did not reach statistical significance.

Conclusion

In the twin model, intra-uterine growth restriction is associated with low-density lipoprotein oxidation without apparent dysregulation of inflammation biomarkers.

Condensation

Increased oxidized low-density lipoproteins are observed in growth restricted twins compared to their co-twins with normal growth at birth.     

Distinctive alterations of invasiveness, drug resistance and cell-cell organization in 3D-cultures of MCF-7, a human breast cancer cell line, and its multidrug resistant variant

Growth of human tumor cells as three-dimensional (3D) multicellular spheroids modifies their invasive properties. Here we study the differences in the biological features of MCF-7, a human breast cancer cell line, and its multidrug resistant variant (MDR-MCF-7) cultured as spheroids or as monolayers. Three-dimensional culture decreased the proliferative rate of both cell lines, reduced the drug sensitivity of MCF-7 cells and did not affect the resistance of MDR-MCF-7 cells. Transmission electron microscopic studies and intercellular junctions labeling showed that MCF-7 spheroids had a junctional system involving E-cadherin, tight-junctions and desmosomes. In MDR-MCF-7 cell spheroids, cell cohesion was mostly due to membrane interdigitations. MDR-MCF-7 cells, but not their parental counterpart, displayed a higher invasive potential when cultured as spheroids, as shown in the Boyden chamber assay. 3D-induced invasiveness was correlated with serine protease and plasminogen activator (PA) secretion. MCF-7 cells did not show any tendency to invade, whatever the mode of culture. These results show that 3D-cultures as spheroids distinctively altered structural features of parental and MDR-MCF-7 cells. In MCF-7 cells, 3D-culture increased cell-cell contacts and drug resistance; in MDR-MCF-7 cells, it induced invasive properties.