The CTLA-4 and PD-1/PD-L1 Inhibitory Pathways Independently Regulate Host Resistance to Plasmodium-induced Acute Immune Pathology

The balance between pro-inflammatory and regulatory immune responses in determining optimal T cell activation is vital for the successful resolution of microbial infections. This balance is maintained in part by the negative regulators of T cell activation, CTLA-4 and PD-1/PD-L, which dampen effector responses during chronic infections. However, their role in acute infections, such as malaria, remains less clear. In this study, we determined the contribution of CTLA-4 and PD-1/PD-L to the regulation of T cell responses during Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM) in susceptible (C57BL/6) and resistant (BALB/c) mice. We found that the expression of CTLA-4 and PD-1 on T cells correlates with the extent of pro-inflammatory responses induced during PbA infection, being higher in C57BL/6 than in BALB/c mice. Thus, ECM develops despite high levels of expression of these inhibitory receptors. However, antibody-mediated blockade of either the CTLA-4 or PD-1/PD-L1, but not the PD-1/PD-L2, pathways during PbA-infection in ECM-resistant BALB/c mice resulted in higher levels of T cell activation, enhanced IFN-γ production, increased intravascular arrest of both parasitised erythrocytes and CD8⁺ T cells to the brain, and augmented incidence of ECM. Thus, in ECM-resistant BALB/c mice, CTLA-4 and PD-1/PD-L1 represent essential, independent and non-redundant pathways for maintaining T cell homeostasis during a virulent malaria infection. Moreover, neutralisation of IFN-γ or depletion of CD8⁺ T cells during PbA infection was shown to reverse the pathologic effects of regulatory pathway blockade, highlighting that the aetiology of ECM in the BALB/c mice is similar to that in C57BL/6 mice. In summary, our results underscore the differential and complex regulation that governs immune responses to malaria parasites.     

The rise of model protozoa

It is timely to evaluate the role of protozoa as model organisms given their diversity, abundance and versatility as well as the economic and ethical pressures placed on animal-based experimentation. We first define the term model organism and then examine through examples why protozoa make good models. Our examples reflect major issues including evolution, ecology, population and community biology, disease, the role of organelles, ageing, space travel, toxicity and teaching. We conclude by recognising that although protozoa may in some cases not completely mimic tissue- or whole-animal-level processes, they are extremely flexible and their use should be embraced. Finally, we offer advice on obtaining emergent model protozoa.     

Harmonine, a defence compound from the harlequin ladybird, inhibits mycobacterial growth and demonstrates multi-stage antimalarial activity

The harlequin ladybird beetle Harmonia axyridis has been introduced in many countries as a biological control agent, but has become an invasive species threatening the biodiversity of native ladybirds. Its invasive success has been attributed to its vigorous resistance against diverse pathogens. This study demonstrates that harmonine ((17R,9Z)-1,17-diaminooctadec-9-ene), which is present in H. axyridis haemolymph, displays broad-spectrum antimicrobial activity that includes human pathogens. Antibacterial activity is most pronounced against fast-growing mycobacteria and Mycobacterium tuberculosis, and the growth of both chloroquine-sensitive and -resistant Plasmodium falciparum strains is inhibited. Harmonine displays gametocytocidal activity, and inhibits the exflagellation of microgametocytes and zygote formation. In an Anopheles stephensi mosquito feeding model, harmonine displays transmission-blocking activity.     

Methane-producing microbial community in a coal bed of the Illinois basin

A series of molecular and geochemical studies were performed to study microbial, coal bed methane formation in the eastern Illinois Basin. Results suggest that organic matter is biodegraded to simple molecules, such as H(2) and CO(2), which fuel methanogenesis and the generation of large coal bed methane reserves. Small-subunit rRNA analysis of both the in situ microbial community and highly purified, methanogenic enrichments indicated that Methanocorpusculum is the dominant genus. Additionally, we characterized this methanogenic microorganism using scanning electron microscopy and distribution of intact polar cell membrane lipids. Phylogenetic studies of coal water samples helped us develop a model of methanogenic biodegradation of macromolecular coal and coal-derived oil by a complex microbial community. Based on enrichments, phylogenetic analyses, and calculated free energies at in situ subsurface conditions for relevant metabolisms (H(2)-utilizing methanogenesis, acetoclastic methanogenesis, and homoacetogenesis), H(2)-utilizing methanogenesis appears to be the dominant terminal process of biodegradation of coal organic matter at this location.     

Lck-dependent Fyn activation requires C terminus-dependent targeting of kinase-active Lck to lipid rafts

Mechanisms regulating the activation and delivery of function of Lck and Fyn are central to the generation of the most proximal signaling events emanating from the T cell antigen receptor (TcR) complex. Recent results demonstrate that lipid rafts (LR) segregate Lck and Fyn and play a fundamental role in the temporal and spatial coordination of their activation. Specifically, TcR-CD4 co-aggregation-induced Lck activation outside LR results in Lck translocation to LR where the activation of LR-resident Fyn ensues. Here we report a structure-function analysis toward characterizing the mechanism supporting Lck partitioning to LR and its capacity to activate co-localized Fyn. Using NIH 3T3 cells ectopically expressing FynT, we demonstrate that only LR-associated, kinase-active (Y505F)Lck reciprocally co-immunoprecipitates with and activates Fyn. Mutational analyses revealed a profound reduction in the formation of Lck-Fyn complexes and Fyn activation, using kinase domain mutants K273R and Y394F of (Y505F)Lck, both of which have profoundly compromised kinase activity. The only kinase-active Lck mutants tested that revealed impaired physical and enzymatic engagement with Fyn were those involving truncation of the C-terminal sequence YQPQP. Remarkably, sequential truncation of YQPQP resulted in an increasing reduction of kinase-active Lck partitioning to LR, in both fibroblasts and T cells. This in turn correlated with an ablation of the capacity of these truncates to enhance TcR-mediated interleukin-2 production. Thus, Lck-dependent Fyn activation is predicated by proximity-mediated transphosphorylation of the Fyn kinase domain, and targeting kinase-active Lck to LR is dependent on the C-terminal sequence QPQP.     

Distinct functions of Cdk5(Y15) phosphorylation and Cdk5 activity in stress fiber formation and organization

Previous studies have shown that Cdk5 promotes lens epithelial cell adhesion. Here we use a cell spreading assay to investigate the mechanism of this effect. As cells spread, forming matrix adhesions and stress fibers, Cdk5(Y15) phosphorylation and Cdk5 kinase activity increased. Cdk5(Y15) phosphorylation was inhibited by PP1, a Src family kinase inhibitor. To identify the PP1-sensitive kinase, we transfected cells with siRNA oligonucleotides for cSrc and related kinases. Only cSrc siRNA oligonucleotides inhibited Cdk5(Y15) phosphorylation. Cdk5(pY15) and its activator, p35, colocalized with actin in stress fibers. To examine Cdk5 function, we inhibited Cdk5 activity under conditions that also prevent phosphorylation at Y15: expression of kinase inactive mutations Cdk5(Y15F) and Cdk5(K33T), and siRNA suppression of Cdk5. Stress fiber formation was severely inhibited. To distinguish between a requirement for Cdk5 kinase activity and a possible adaptor role for Cdk5(pY15), we used two methods that inhibit kinase activity without inhibiting phosphorylation at Y15: pharmacological inhibition with olomoucine and expression of the kinase inactive mutation, Cdk5(D144N). Stress fiber organization was altered, but stress fiber formation was not blocked. These findings indicate that Cdk5(Y15) phosphorylation and Cdk5 activity have distinct functions required for stress fiber formation and organization, respectively.     

Functional Properties of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strains Isolated from Maasai Traditional Fermented Milk Products in Kenya

Lactobacillus plantarum was the major species among the lactic acid bacterial strains isolated from traditional fermented milk of the Maasai in Kenya. Selected strains were characterized for their functional properties using in vitro standard procedures. All strains expressed acid tolerance at pH 2.0 after 2-h exposure of values that ranged from 1% to 100%, while bile tolerance of acid-stressed cells at 0.3% oxgal varied from 30% to 80%. In vitro adhesion to the mucus-secreting cell line HT 29 MTX and binding capacity to extracellular protein matrices was demonstrated for several strains. The four strains tested in a simulated stomach duodenum passage survived with recovery rates ranging from 17% to 100%. Strains were intrinsically resistant to several antibiotics tested. From these in vitro studies, a number of Lb. plantarum strains isolated from the Maasai traditional fermented milk showed probiotic potential. The strains are good candidates for multifunctional starter culture development.     

A homogeneous cellular histone deacetylase assay suitable for compound profiling and robotic screening

Most cellular assays that quantify the efficacy of histone deacetylase (HDAC) inhibitors measure hyperacetylation of core histone proteins H3 and H4. Here we describe a new approach, directly measuring cellular HDAC enzymatic activity using the substrate Boc-K(Ac)-7-amino-4-methylcoumarin (AMC). After penetration into HeLa cervical carcinoma or K562 chronic myeloid leukemia cells, the deacetylated product Boc-K-AMC is formed which, after cell lysis, is cleaved by trypsin, finally releasing the fluorophor AMC. The cellular potency of suberoylanilide hydroxamic acid, LBH589, trichostatin A, and MS275 as well-known HDAC inhibitors was determined using this assay. IC(50) values derived from concentration-effect curves correlated well with EC(50) values derived from a cellomics array scan histone H3 hyperacetylation assay. The cellular HDAC activity assay was adapted to a homogeneous format, fully compatible with robotic screening. Concentration-effect curves generated on a Tecan Genesis Freedom workstation were highly reproducible with a signal-to-noise ratio of 5.7 and a Z' factor of 0.88, indicating a very robust assay. Finally, a HDAC-inhibitor focused library was profiled in a medium-throughput screening campaign. Inhibition of cellular HDAC activity correlated well with cytotoxicity and histone H3 hyperacetylation in HeLa cells and with inhibition of human recombinant HDAC1 in a biochemical assay. Thus, by using Boc-K(Ac)-AMC as a cell-permeable HDAC substrate, the activity of various protein lysine-specific deacetylases including HDAC1-containing complexes is measurable in intact cells in a simple and homogeneous manner.