In vivo imaging and quantitative analysis of leukocyte directional migration and polarization in inflamed tissue

Directional migration of transmigrated leukocytes to the site of injury is a central event in the inflammatory response. Here, we present an in vivo chemotaxis assay enabling the visualization and quantitative analysis of subtype-specific directional motility and polarization of leukocytes in their natural 3D microenvironment. Our technique comprises the combination of i) semi-automated in situ microinjection of chemoattractants or bacteria as local chemotactic stimulus, ii) in vivo near-infrared reflected-light oblique transillumination (RLOT) microscopy for the visualization of leukocyte motility and morphology, and iii) in vivo fluorescence microscopy for the visualization of different leukocyte subpopulations or fluorescence-labeled bacteria. Leukocyte motility parameters are quantified off-line in digitized video sequences using computer-assisted single cell tracking. Here, we show that perivenular microinjection of chemoattractants [macrophage inflammatory protein-1alpha (MIP-1alpha/Ccl3), platelet-activating factor (PAF)] or E. coli into the murine cremaster muscle induces target-oriented intravascular adhesion and transmigration as well as polarization and directional interstitial migration of leukocytes towards the locally administered stimuli. Moreover, we describe a crucial role of Rho kinase for the regulation of directional motility and polarization of transmigrated leukocytes in vivo. Finally, combining in vivo RLOT and fluorescence microscopy in Cx3CR1(gfp/gfp) mice (mice exhibiting green fluorescent protein-labeled monocytes), we are able to demonstrate differences in the migratory behavior of monocytes and neutrophils.Taken together, we propose a novel approach for investigating the mechanisms and spatiotemporal dynamics of subtype-specific motility and polarization of leukocytes during their directional interstitial migration in vivo

Complete mitochondrial DNA sequences provide new insights into the Polynesian motif and the peopling of Madagascar

More than a decade of mitochondrial DNA (mtDNA) studies have given the 'Polynesian motif' renowned status as a marker for tracing the late-Holocene expansion of Austronesian speaking populations. Despite considerable research on the Polynesian motif in Oceania, there has been little equivalent work on the western edge of its expansion - leaving major issues unresolved regarding the motif's evolutionary history. This has also led to considerable uncertainty regarding the settlement of Madagascar. In this study, we assess mtDNA variation in 266 individuals from three Malagasy ethnic groups: the Mikea, Vezo, and Merina. Complete mtDNA genome sequencing reveals a new variant of the Polynesian motif in Madagascar; two coding region mutations define a Malagasy-specific sub-branch. This newly defined 'Malagasy motif' occurs at high frequency in all three ethnic groups (13-50%), and its phylogenetic position, geographic distribution, and estimated age all support a recent origin, but without conclusively identifying a specific source region. Nevertheless, the haplotype's limited diversity, similar to those of other mtDNA haplogroups found in our Malagasy groups, best supports a small number of initial settlers arriving to Madagascar through the same migratory process. Finally, the discovery of this lineage provides a set of new polymorphic positions to help localize the Austronesian ancestors of the Malagasy, as well as uncover the origin and evolution of the Polynesian motif itself

Persistent High Burden of Invasive Pneumococcal Disease in South African HIV-Infected Adults in the Era of an Antiretroviral Treatment Program

Highly active antiretroviral treatment (HAART) programs have been associated with declines in the burden of invasive pneumococcal disease (IPD) in industrialized countries. The aim of this study was to evaluate trends in IPD hospitalizations in HIV-infected adults in Soweto, South Africa, associated with up-scaling of the HAART program from 2003 to 2008.Laboratory-confirmed IPD cases were identified from 2003 through 2008 through an existing surveillance program. The period 2003-04 was designated as the early-HAART era, 2005-06 as the intermediate-HAART era and 2007-08 as the established-HAART era. The incidence of IPD was compared between the early-HAART and established-HAART eras in HIV-infected and-uninfected individuals.A total of 2,567 IPD cases among individuals older than 18 years were reported from 2003 through 2008. Overall incidence of IPD (per 100,000) did not change during the study period in HIV-infected adults (207.4 cases in the early-HAART and 214.0 cases in the established-HAART era; p = 0.55). IPD incidence, actually increased 1.16-fold (95% CI: 1.01; 1.62) in HIV-infected females between the early-and established-HAART eras (212.1 cases and 246.2 cases, respectively; p = 0.03). The incidence of IPD remained unchanged in HIV-uninfected adults across the three time periods.Despite a stable prevalence of HIV and the increased roll-out of HAART for treatment of AIDS patients in our setting, the burden of IPD has not decreased among HIV-infected adults. The study indicates a need for ongoing monitoring of disease and HAART program effectiveness to reduce opportunistic infections in African adults with HIV/AIDS, as well as the need to consider alternate strategies including pneumococcal conjugate vaccine immunization for the prevention of IPD in HIV-infected adults

Predictive Value of Fever and Palmar Pallor for P. falciparum Parasitaemia in Children from an Endemic Area

INTRODUCTION: Although the incidence of Plasmodium falciparum malaria in some parts of sub-Saharan Africa is reported to decline and other conditions, causing similar symptoms as clinical malaria are gaining in relevance, presumptive anti-malarial treatment is still common. This study traced for age-dependent signs and symptoms predictive for P. falciparum parasitaemia. METHODS: In total, 5447 visits of 3641 patients between 2-60 months of age who attended an outpatient department (OPD) of a rural hospital in the Ashanti Region, Ghana, were analysed. All Children were examined by a paediatrician and a full blood count and thick smear were done. A Classification and Regression Tree (CART) model was used to generate a clinical decision tree to predict malarial parasitaemia a7nd predictive values of all symptoms were calculated. RESULTS: Malarial parasitaemia was detected in children between 2-12 months and between 12-60 months of age with a prevalence of 13.8% and 30.6%, respectively. The CART-model revealed age-dependent differences in the ability of the variables to predict parasitaemia. While palmar pallor was the most important symptom in children between 2-12 months, a report of fever and an elevated body temperature of ≥37.5°C gained in relevance in children between 12-60 months. The variable palmar pallor was significantly (p<0.001) associated with lower haemoglobin levels in children of all ages. Compared to the Integrated Management of Childhood Illness (IMCI) algorithm the CART-model had much lower sensitivities, but higher specificities and positive predictive values for a malarial parasitaemia. CONCLUSIONS: Use of age-derived algorithms increases the specificity of the prediction for P. falciparum parasitaemia. The predictive value of palmar pallor should be underlined in health worker training. Due to a lack of sensitivity neither the best algorithm nor palmar pallor as a single sign are eligible for decision-making and cannot replace presumptive treatment or laboratory diagnosis

Working without a blindfold: the critical role of diagnostics in malaria control

Diagnostic testing for malaria has for many years been eschewed, lest it be an obstacle to the delivery of rapid, life-saving treatment. The approach of treating malaria without confirmatory testing has been reinforced by the availability of inexpensive treatment with few side effects, by the great difficulty of establishing quality-assured microscopy in rural and resource-poor settings, and by the preeminence of malaria as a cause of important fever in endemic regions. Within the last decade, all three of these factors have changed. More expensive artemisinin combination therapy (ACT) has been widely introduced, simple immunochromatographic tests for malaria have been developed that can be used as an alternative to microscopy by village health workers, and recognition of the health cost of mismanaging non-malarial fever is growing. In most of the world a small fraction of fever is due to malaria, and reflex treatment with ACT does not make medical or economic sense. Global malaria control efforts have been energized by the availability of new sources of funding, and by the rapid reduction in malaria prevalence in a number of settings where bed nets, indoor residual spraying with insecticides, and ACT have been systematically deployed. This momentum has been captured by a new call for malaria elimination. Without wide implementation of accurate and discriminating diagnostic testing, and reporting of results, most fever will be inappropriately managed, millions of doses of ACT will be wasted, and malaria control programmes will be blindfolded to the impact of their efforts

Prospective study on severe malaria among in-patients at Bombo regional hospital, Tanga, north-eastern Tanzania

In Tanzania, malaria is the major cause of morbidity and mortality, accounting for about 30% of all hospital admissions and around 15% of all hospital deaths. Severe anaemia and cerebral malaria are the two main causes of death due to malaria in Tanga, Tanzania. This was a prospective observational hospital-based study conducted from October 2004 to September 2005. Consent was sought from study participants or guardians in the wards. Finger prick blood was collected from each individual for thick and thin smears, blood sugar levels and haemoglobin estimations by Haemocue machine after admission. A total of 494 patients were clinically diagnosed and admitted as cases of severe malaria. Majority of them (55.3%) were children below the age of 5 years. Only 285 out of the total 494 (57.7%) patients had positive blood smears for malaria parasites. Adults aged 20 years and above had the highest rate of cases with fever and blood smear negative for malaria parasites. Commonest clinical manifestations of severe malaria were cerebral malaria (47.3%) and severe anaemia (14.6%), particularly in the under-fives. Case fatality was 3.2% and majority of the deaths occurred in the under-fives and adults aged 20 years and above with negative blood smears. Proper laboratory diagnosis is crucial for case management and reliable data collection. The non-specific nature of malaria symptomatologies limits the use of clinical diagnosis and the IMCI strategy. Strengthening of laboratory investigations to guide case management is recommended

Morphological characterization of bushy cells and their inputs in the laboratory mouse (Mus musculus) anteroventral cochlear nucleus.

PMC3753269Spherical and globular bushy cells of the AVCN receive huge auditory nerve endings specialized for high fidelity neural transmission in response to acoustic events. Recent studies in mice and other rodent species suggest that the distinction between bushy cell subtypes is not always straightforward. We conducted a systematic investigation of mouse bushy cells along the rostral-caudal axis in an effort to understand the morphological variation that gives rise to reported response properties in mice. We combined quantitative light and electron microscopy to investigate variations in cell morphology, immunostaining, and the distribution of primary and non-primary synaptic inputs along the rostral-caudal axis. Overall, large regional differences in bushy cell characteristics were not found; however, rostral bushy cells received a different complement of axosomatic input compared to caudal bushy cells. The percentage of primary auditory nerve terminals was larger in caudal AVCN, whereas non-primary excitatory and inhibitory inputs were more common in rostral AVCN. Other ultrastructural characteristics of primary auditory nerve inputs were similar across the rostral and caudal AVCN. Cross sectional area, postsynaptic density length and curvature, and mitochondrial volume fraction were similar for axosomatic auditory nerve terminals, although rostral auditory nerve terminals contained a greater concentration of synaptic vesicles near the postsynaptic densities. These data demonstrate regional differences in synaptic organization of inputs to mouse bushy cells rather than the morphological characteristic of the cells themselves.JH Libraries Open Access Fun

The HIV-1 transmission bottleneck

It is well established that most new systemic infections of HIV-1 can be traced back to one or a limited number of founder viruses. Usually, these founders are more closely related to minor HIV-1 populations in the blood of the presumed donor than to more abundant lineages. This has led to the widely accepted idea that transmission selects for viral characteristics that facilitate crossing the mucosal barrier of the recipient’s genital tract, although the specific selective forces or advantages are not completely defined. However, there are other steps along the way to becoming a founder virus at which selection may occur. These steps include the transition from the donor’s general circulation to the genital tract compartment, survival within the transmission fluid, and establishment of a nascent stable local infection in the recipient’s genital tract. Finally, there is the possibility that important narrowing events may also occur during establishment of systemic infection. This is suggested by the surprising observation that the number of founder viruses detected after transmission in intravenous drug users is also limited. Although some of these steps may be heavily selective, others may result mostly in a stochastic narrowing of the available founder pool. Collectively, they shape the initial infection in each recipient