Transient Synaptic Enhancement Triggered by Exogenously Supplied Monocarboxylate in Drosophila Motoneuron Synapse.

Current evidence suggests that glial cells provide C3 carbon sources to fuel neuronal activity; however, this notion has become challenged by biosensor studies carried out in acute brain slices or in vivo, showing that neuronal activity does not rely on the import of astrocyte-produced L-lactate. Rather, stimulated neurons become net lactate exporters, as it was also shown in Drosophila neurons, in which astrocyte-provided lactate returns as lipid droplets to be stored in glial cells. In this view, we investigate whether exogenously supplied monocarboxylates can support Drosophila motoneuron neurotransmitter release (NTR). By assessing the excitatory post-synaptic current (EPSC) amplitude under voltage-clamp as NTR indicative, we found that both pyruvate and L-lactate, as the only carbon sources in the synapses bathing-solution, cause a large transient NTR enhancement, which declines to reach a synaptic depression state, from which the synapses do not recover. The FM1-43 pre-synaptic loading ability, however, is maintained under monocarboxylate, suggesting that SV cycling should not contribute to the synaptic depression state. The NTR recovery was reached by supplementing the monocarboxylate medium with sucrose. However, monocarboxylate addition to sucrose medium does not enhance NTR, but it does when the disaccharide concentration becomes too reduced. Thus, when pyruvate concentrations become too reduced, exogenously supplied L-lactate could be converted to pyruvate and metabolized by the neural mitochondria, triggering the NTR enhancement. SIGNIFICANCE STATEMENT: The question of whether monocarboxylic acids can fuel the Drosophila motoneuron NTR was challenged. Our findings show that exogenously supplied monocarboxylates trigger a large transient synaptic enhancement just under extreme glycolysis reduction but fail to maintain NTR under sustained synaptic demand, still at low frequency stimulation, driven to the synapses to a synaptic depression state. Glycolysis activation, by adding sucrose to the monocarboxylate bath solution, restores the motoneuron NTR ability, giving place to a hexoses role in SV recruitment. Moreover these results suggest exogenously supplied C3 carbon sources could have an additional role beyond providing energetic support for neural activity.

RUFY3 regulates endolysosomes perinuclear positioning, antigen presentation and migration in activated phagocytes.

Endo-lysosomes transport along microtubules and clustering in the perinuclear area are two necessary steps for microbes to activate specialized phagocyte functions. We report that RUN and FYVE domain-containing protein 3 (RUFY3) exists as two alternative isoforms distinguishable by the presence of a C-terminal FYVE domain and by their affinity for phosphatidylinositol 3-phosphate on endosomal membranes. The FYVE domain-bearing isoform (iRUFY3) is preferentially expressed in primary immune cells and up-regulated upon activation by microbes and Interferons. iRUFY3 is necessary for ARL8b + /LAMP1+ endo-lysosomes positioning in the pericentriolar organelles cloud of LPS-activated macrophages. We show that iRUFY3 controls macrophages migration, MHC II presentation and responses to Interferon-γ, while being important for intracellular Salmonella replication. Specific inactivation of rufy3 in phagocytes leads to aggravated pathologies in mouse upon LPS injection or bacterial pneumonia. This study highlights the role of iRUFY3 in controlling endo-lysosomal dynamics, which contributes to phagocyte activation and immune response regulation.

How cell migration helps immune sentinels.

The immune system relies on the migratory capacity of its cellular components, which must be mobile in order to defend the host from invading micro-organisms or malignant cells. This applies in particular to immune sentinels from the myeloid lineage, i.e. macrophages and dendritic cells. Cell migration is already at work during mammalian early development, when myeloid cell precursors migrate from the yolk sac, an extra embryonic structure, to colonize tissues and form the pool of tissue-resident macrophages. Later, this is accompanied by a migration wave of precursors and monocytes from the bone marrow to secondary lymphoid organs and the peripheral tissues. They differentiate into DCs and monocyte-derived macrophages. During adult life, cell migration endows immune cells with the ability to patrol their environment as well as to circulate between peripheral tissues and lymphoid organs. Hence migration of immune cells is key to building an efficient defense system for an organism. In this review, we will describe how cell migratory capacity regulates the various stages in the life of myeloid cells from development to tissue patrolling, and migration to lymph nodes. We will focus on the role of the actin cytoskeletal machinery and its regulators, and how it contributes to the establishment and function of the immune system.

Macropinocytosis and Cell Migration: Don't Drink and Drive .

Macropinocytosis is a nonspecific mechanism by which cells compulsively "drink" the surrounding extracellular fluids in order to feed themselves or sample the molecules therein, hence gaining information about their environment. This process is cell-intrinsically incompatible with the migration of many cells, implying that the two functions are antagonistic. The migrating cell uses a molecular switch to stop and explore its surrounding fluid by macropinocytosis, after which it employs the same molecular machinery to start migrating again to examine another location. This cycle of migration/macropinocytosis allows cells to explore tissues, and it is key to a range of physiological processes. Evidence of this evolutionarily conserved antagonism between the two processes can be found in several cell types-immune cells, for example, being particularly adept-and ancient organisms (e.g., the social amoeba Dictyostelium discoideum). How macropinocytosis and migration are negatively coupled is the subject of this chapter.

Epithelial colonization by gut dendritic cells promotes their functional diversification.

Dendritic cells (DCs) patrol tissues and transport antigens to lymph nodes to initiate adaptive immune responses. Within tissues, DCs constitute a complex cell population composed of distinct subsets that can exhibit different activation states and functions. How tissue-specific cues orchestrate DC diversification remains elusive. Here, we show that the small intestine included two pools of cDC2s originating from common pre-DC precursors: (1) lamina propria (LP) CD103+CD11b+ cDC2s that were mature-like proinflammatory cells and (2) intraepithelial cDC2s that exhibited an immature-like phenotype as well as tolerogenic properties. These phenotypes resulted from the action of food-derived retinoic acid (ATRA), which enhanced actomyosin contractility and promoted LP cDC2 transmigration into the epithelium. There, cDC2s were imprinted by environmental cues, including ATRA itself and the mucus component Muc2. Hence, by reaching distinct subtissular niches, DCs can exist as immature and mature cells within the same tissue, revealing an additional mechanism of DC functional diversification.

Macropinocytosis Overcomes Directional Bias in Dendritic Cells Due to Hydraulic Resistance and Facilitates Space Exploration.

The migration of immune cells can be guided by physical cues imposed by the environment, such as geometry, rigidity, or hydraulic resistance (HR). Neutrophils preferentially follow paths of least HR in vitro, a phenomenon known as barotaxis. The mechanisms and physiological relevance of barotaxis remain unclear. We show that barotaxis results from the amplification of a small force imbalance by the actomyosin cytoskeleton, resulting in biased directional choices. In immature dendritic cells (DCs), actomyosin is recruited to the cell front to build macropinosomes. These cells are therefore insensitive to HR, as macropinocytosis allows fluid transport across these cells. This may enhance their space exploration capacity in vivo. Conversely, mature DCs down-regulate macropinocytosis and are thus barotactic. Modeling suggests that HR may help guide these cells to lymph nodes where they initiate immune responses. Hence, DCs can either overcome or capitalize on the physical obstacles they encounter, helping their immune-surveillance function.

Light-Induced Opening of the TRP Channel in Isolated Membrane Patches Excised from Photosensitive Microvilli from Drosophila Photoreceptors.

Drosophila phototransduction occurs in light-sensitive microvilli arranged in a longitudinal structure of the photoreceptor, termed the rhabdomere. Rhodopsin (Rh), isomerized by light, couples to G-protein, which activates phospholipase C (PLC), which in turn cleaves phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG), inositol trisphosphate and H+. This pathway opens the light-dependent channels, transient receptor potential (TRP) and transient receptor potential like (TRPL). PLC and TRP are held together in a protein assembly by the scaffold protein INAD. We report that the channels can be photoactivated in on-cell rhabdomeric patches and in excised patches by DAG. In excised patches, addition of PLC-activator, m-3M3FBS, or G-protein-activator, GTP-γ-S, opened TRP. These reagents were ineffective in PLC-mutant norpA and in the presence of PLC inhibitor U17322. However, DAG activated TRP even when PLC was pharmacologically or mutationally suppressed. These observations indicate that PLC, G-protein, and TRP were retained functional in these patches. DAG also activated TRP in the protein kinase C (PKC) mutant, inaC, excluding the possibility that PKC could mediate DAG-dependent TRP activation. Labeling diacylglycerol kinase (DGK) by fusion of fluorescent mCherry (mCherry-DGK) indicates that DGK, which returns DAG to dark levels, is highly expressed in the microvilli. In excised patches, TRP channels could be light-activated in the presence of GTP, which is required for G-protein activation. The evidence indicates that the proteins necessary for phototransduction are retained functionally after excision and that DAG is necessary and sufficient for TRP opening. This work opens up unique possibilities for studying, in sub-microscopic native membrane patches, the ubiquitous phosphoinositide signaling pathway and its regulatory mechanisms in unprecedented detail.

An in vitro method for studying subcellular rearrangements during cell polarization in Drosophila melanogaster hemocytes.

Thanks to the power of Drosophila genetics, this animal model has been a precious tool for scientists to uncover key processes associated to innate immunity. The fly immune system relies on a population of macrophage-like cells, also referred to as hemocytes, which are highly migratory and phagocytic, and can easily be followed in vivo. These cells have shown to play important roles in fly development, both at the embryonic and pupal stages. However, there is no robust assay for the study of hemocyte migration in vitro, which limits our understanding of the molecular mechanisms involved. Here, we contribute to fill this gap by showing that hemocytes adopt a polarized morphology upon ecdysone stimulation, allowing the study of the cytoskeleton rearrangements and organelle reorganization that take place during the first step of cell locomotion.

Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress.

The intercellular transport of lactate is crucial for the astrocyte-to-neuron lactate shuttle (ANLS), a model of brain energetics according to which neurons are fueled by astrocytic lactate. In this study we show that the Drosophila chaski gene encodes a monocarboxylate transporter protein (MCT/SLC16A) which functions as a lactate/pyruvate transporter, as demonstrated by heterologous expression in mammalian cell culture using a genetically encoded FRET nanosensor. chaski expression is prominent in the Drosophila central nervous system and it is particularly enriched in glia over neurons. chaski mutants exhibit defects in a high energy demanding process such as synaptic transmission, as well as in locomotion and survival under nutritional stress. Remarkably, locomotion and survival under nutritional stress defects are restored by chaski expression in glia cells. Our findings are consistent with a major role for intercellular lactate shuttling in the brain metabolism of Drosophila.

A CLCA regulatory protein present in the chemosensory cilia of olfactory sensory neurons induces a Ca2+-activated Cl- current when transfected into HEK293.

BACKGROUND: CLCA is a family of metalloproteases that regulate Ca2+-activated Cl- fluxes in epithelial tissues. In HEK293 cells, CLCA1 promotes membrane expression of an endogenous Anoctamin 1 (ANO1, also termed TMEM16A)-dependent Ca2+-activated Cl- current. Motif architecture similarity with CLCA2, 3 and 4 suggested that they have similar functions. We previously detected the isoform CLCA4L in rat olfactory sensory neurons, where Anoctamin 2 is the principal chemotransduction Ca2+-activated Cl- channel. We explored the possibility that this protein plays a role in odor transduction. RESULTS: We cloned and expressed CLCA4L from rat olfactory epithelium in HEK293 cells. In the transfected HEK293 cells we measured a Cl--selective Ca2+-activated current, blocked by niflumic acid, not present in the non-transfected cells. Thus, CLCA4L mimics the CLCA1 current on its ability to induce the ANO1-dependent Ca2+-activated Cl- current endogenous to these cells. By immunocytochemistry, a CLCA protein, presumably CLCA4L, was detected in the cilia of olfactory sensory neurons co-expressing with ANO2. CONCLUSION: These findings suggests that a CLCA isoform, namely CLCA4L, expressed in OSN cilia, might have a regulatory function over the ANO2-dependent Ca2+-activated Cl- channel involved in odor transduction.

Vitamin A supplements, routine immunization, and the subsequent risk of Plasmodium infection among children under 5 years in sub-Saharan Africa.

Recent studies, partly based on murine models, suggest childhood immunization and vitamin A supplements may confer protection against malaria infection, although strong evidence to support these theories in humans has so far been lacking. We analyzed national survey data from children aged 6-59 months in four sub-Saharan African countries over an 18-month time period, to determine the risk of Plasmodium spp. parasitemia (n=8390) and Plasmodium falciparum HRP-2 (PfHRP-2)-related antigenemia (n=6121) following vitamin A supplementation and standard vaccination. Bacille Calmette Guerin-vaccinated children were more likely to be PfHRP-2 positive (relative risk [RR]=4.06, 95% confidence interval [CI]=2.00-8.28). No association was identified with parasitemia. Measles and polio vaccination were not associated with malaria. Children receiving vitamin A were less likely to present with parasitemia (RR=0.46, 95% CI=0.39-0.54) and antigenemia (RR=0.23, 95% CI=0.17-0.29). Future studies focusing on climate seasonality, placental malaria and HIV are needed to characterize better the association between vitamin A and malaria infection in different settings.

Acute lower respiratory infection among Bacille Calmette-Guérin (BCG)-vaccinated children.

OBJECTIVE: To determine whether Bacille Calmette-Guerin (BCG) vaccination is linked to the risk of acute lower respiratory infection (ALRI) among children <5 years of age. METHODS: Data from Macro International Demographic and Health Surveys and United Nations Children's Fund Multiple Indicator Cluster Surveys were used to identify a primary cohort of 58,021 children in 19 countries (2005-2010) and a secondary cohort of 93,301 children in 18 countries (2000-2007). Information was collected by trained interviewers during home visits using standardized questionnaires, review of vaccination health cards, and measurement of health indicators. RESULTS: BCG vaccination was associated with a 17% to 37% risk reduction for suspected ALRI in both cohorts. The only vaccine or vitamin supplement to modify the effect of BCG was diphtheria-tetanus-pertussis (DTP; P < .001). The order in which the vaccines were first received was central to this phenomena (BCG before DTP, adjusted/propensity score-weighted relative risk [apRR]: 0.79, 95% confidence interval [CI]: 0.70-0.89; BCG with DTP, apRR: 0.82, 95% CI: 0.71-0.94; and BCG after DTP, apRR: 1.00, 95% CI: 0.87-1.13) but not number of DTP doses received. Other modifiers included vaccine strain used in immunization programs, chlorinating drinking water, using wood-burning fuel cook stoves, and owning livestock. CONCLUSIONS: Children vaccinated with BCG had a significantly lower risk of suspected ALRI. Clarification is needed as to whether this is due to reductions in the underlying risk of tuberculosis or ALRI per se.

Resistance to fluoroquinolones and second-line injectable drugs: impact on multidrug-resistant TB outcomes.

A meta-analysis for response to treatment was undertaken using individual data of multidrug-resistant tuberculosis (MDR-TB) (resistance to isoniazid and rifampicin) patients from 26 centres. The analysis assessed the impact of additional resistance to fluoroquinolones and/or second-line injectable drugs on treatment outcome. Compared with treatment failure, relapse and death, treatment success was higher in MDR-TB patients infected with strains without additional resistance (n=4763; 64%, 95% CI 57-72%) or with resistance to second-line injectable drugs only (n=1130; 56%, 95% CI 45-66%), than in those having resistance to fluoroquinolones alone (n=426; 48%, 95% CI 36-60%) or to fluoroquinolones plus second-line injectable drugs (extensively drug resistant (XDR)-TB) (n=405; 40%, 95% CI 27-53%). In XDR-TB patients, treatment success was highest if at least six drugs were used in the intensive phase (adjusted OR 4.9, 95% CI 1.4-16.6; reference fewer than three drugs) and four in the continuation phase (OR 6.1, 95% CI 1.4-26.3). The odds of success in XDR-TB patients was maximised when the intensive phase reached 6.6-9.0 months duration and the total duration of treatment 20.1-25.0 months. In XDR-TB patients, regimens containing more drugs than those recommended in MDR-TB but given for a similar duration were associated with the highest odds of success. All data were from observational studies and methodologies varied between centres, therefore, the bias may be substantial. Better quality evidence is needed to optimise regimens.

Molecular epidemiology of tuberculosis transmission: Contextualizing the evidence through social network theory.

Despite a long-standing recognition that factors such as age, gender, and socioeconomic status play a fundamental role in tuberculosis transmission and susceptibility, few molecular epidemiological studies have fully elucidated the etiological mechanisms by which each of these social factors may influence transmission of the disease. In this paper, we propose that in order to achieve this goal, molecular epidemiology must move towards a more holistic approach for disease transmission, thus enabling social theory to be integrated into molecular epidemiological studies on tuberculosis. We then present a social network model to illustrate how molecular and social epidemiology can be combined to study disease transmission patterns, and provide preliminary molecular epidemiological evidence to support the role of social networks in tuberculosis transmission.

Lack of an adverse effect of Giardia intestinalis infection on the health of Peruvian children.

Giardia intestinalis is a common gastrointestinal protozoan worldwide, but its effects on childhood growth in developing countries are not clearly understood. The authors aimed to describe its effects on child growth. They followed 220 Peruvian children daily for diarrhea, weekly for stool samples, and monthly for anthropometry. The authors modeled the effect of nutritional status on the risk of Giardia infection and the risk of diarrhea attributable to Giardia using negative binomial regression. They modeled the effects of Giardia infection on growth using linear regression, with 85% of children becoming infected with Giardia and 87% of these becoming reinfected. In multivariable analysis, the risk of Giardia infection did not vary with weight for age (relative risk = 1.00, 95% confidence interval: 0.89, 1.12) or height for age (relative risk = 0.92, 95% confidence interval: 0.82, 1.04). Giardiasis did not affect growth at 1 or 2 months following the first infection at any age interval. The longitudinal prevalence of Giardia between 6 and 24 months of age was not associated with height gain in that interval (p = 0.981). Giardia was not associated with an increased risk of diarrhea at any age interval. Study results question the importance of Giardia as a childhood pathogen in developing countries where giardiasis is hyperendemic.

Scaffolding proteins in highly purified rat olfactory cilia membranes.

Odour-mediated signal transduction is a complex process that occurs in the cilia of olfactory sensory neurons. To gain insight in to the molecular organization of the odour transduction machinery, we developed a procedure to purify olfactory cilia membranes by differential centrifugation of rat olfactory epithelium extracts. We tested whether known scaffolding proteins that might participate in the assembly of the complex chemotransduction apparatus are present in the purified membrane fraction. Utilizing immunoblotting and immunohistochemistry, we show that the multidomain scaffolding proteins ProSAP/Shanks and calcium/calmodulin-dependent serine protein kinase CASK are present in the olfactory cilia. Ion channels involved in chemotransduction could be reconstituted into planar lipid bilayers for electrophysiological recordings. Our procedure should allow the identification of further chemotransduction-related proteins.

Microscopic-observation drug-susceptibility assay for the diagnosis of TB.

BACKGROUND: New diagnostic tools are urgently needed to interrupt the transmission of tuberculosis and multidrug-resistant tuberculosis. Rapid, sensitive detection of tuberculosis and multidrug-resistant tuberculosis in sputum has been demonstrated in proof-of-principle studies of the microscopic-observation drug-susceptibility (MODS) assay, in which broth cultures are examined microscopically to detect characteristic growth. METHODS: In an operational setting in Peru, we investigated the performance of the MODS assay for culture and drug-susceptibility testing in three target groups: unselected patients with suspected tuberculosis, prescreened patients at high risk for tuberculosis or multidrug-resistant tuberculosis, and unselected hospitalized patients infected with the human immunodeficiency virus. We compared the MODS assay head-to-head with two reference methods: automated mycobacterial culture and culture on Löwenstein-Jensen medium with the proportion method. RESULTS: Of 3760 sputum samples, 401 (10.7%) yielded cultures positive for Mycobacterium tuberculosis. Sensitivity of detection was 97.8% for MODS culture, 89.0% for automated mycobacterial culture, and 84.0% for Löwenstein-Jensen culture (P<0.001); the median time to culture positivity was 7 days, 13 days, and 26 days, respectively (P<0.001), and the median time to the results of susceptibility tests was 7 days, 22 days, and 68 days, respectively. The incremental benefit of a second MODS culture was minimal, particularly in patients at high risk for tuberculosis or multidrug-resistant tuberculosis. Agreement between MODS and the reference standard for susceptibility was 100% for rifampin, 97% for isoniazid, 99% for rifampin and isoniazid (combined results for multidrug resistance), 95% for ethambutol, and 92% for streptomycin (kappa values, 1.0, 0.89, 0.93, 0.71, and 0.72, respectively). CONCLUSIONS: A single MODS culture of a sputum sample offers more rapid and sensitive detection of tuberculosis and multidrug-resistant tuberculosis than the existing gold-standard methods used.

Infrequent MODS TB culture cross-contamination in a high-burden resource-poor setting.

One obstacle to wider use of rapid liquid culture-based tuberculosis diagnostics such as the microscopic observation drug susceptibility (MODS) assay is concern about cross-contamination. We investigated the rate of laboratory cross-contamination in MODS, automated MBBacT, and Lowenstein-Jensen (LJ) cultures performed in parallel, through triangulation of microbiologic (reculturing stored samples), molecular (spoligotype/RFLP), and clinical epidemiologic data. At least 1 culture was positive for Mycobacterium tuberculosis for 362 (11%) of 3416 samples; 53 were regarded as potential cross-contamination suspects. Cross-contamination accounted for 17 false-positive cultures from 14 samples representing 0.41% (14/3416) and 0.17% (17/10248) of samples and cultures, respectively. Positive predictive values for MODS, MBBacT (bioMérieux, Durham, NC), and LJ were 99.1%, 98.7%, and 99.7%, and specificity was 99.9% for all 3. Low rates of cross-contamination are achievable in mycobacterial laboratories in resource-poor settings even when a large proportion of samples are infectious and highly sensitive liquid culture-based diagnostics such as MODS are used.

Invasive group A streptococcal infections, clinical manifestations and their predictors, Montreal, 1995-2001.

We identified 306 invasive group A streptococcal infections (IGASI) by passive population-based surveillance in Montreal, Canada, from 1995 to 2001. The average yearly reported incidence was 2.4 per 100,000 persons, with a 14% death rate. Among clinical manifestations, incidence of pneumonia increased from 0.06 per 100,000 in 1995 to 0.50 per 100,000 in 2000. Over a span of 7 years, the odds of developing pneumonia increased (odds ratio [OR] = 1.21, 95% confidence interval [CI] 1.0-1.5), while they decreased for soft-tissue infections (OR = 0.86, 95% CI 0.7-1.0). Serotypes M1 and M3 accounted for 30% of IGASI. However, neither serotype was significantly associated with specific clinical manifestations, which suggests that manifestation development among IGASI might be attributable to host or environmental factors rather than the pathogen. In our study, these factors included age, gender, underlying medical conditions, and living environment, yet none explained temporal changes in risk for pneumonia and soft-tissue infections.