Protective immunity induced with the RTS,S/AS vaccine is associated with IL-2 and TNF-α producing effector and central memory CD4 T cells

A phase 2a RTS,S/AS malaria vaccine trial, conducted previously at the Walter Reed Army Institute of Research, conferred sterile immunity against a primary challenge with infectious sporozoites in 40% of the 80 subjects enrolled in the study. The frequency of Plasmodium falciparum circumsporozoite protein (CSP)-specific CD4(+) T cells was significantly higher in protected subjects as compared to non-protected subjects. Intrigued by these unique vaccine-related correlates of protection, in the present study we asked whether RTS,S also induced effector/effector memory (T(E/EM)) and/or central memory (T(CM)) CD4(+) T cells and whether one or both of these sub-populations is the primary source of cytokine production. We showed for the first time that PBMC from malaria-non-exposed RTS,S-immunized subjects contain both T(E/EM) and T(CM) cells that generate strong IL-2 responses following re-stimulation in vitro with CSP peptides. Moreover, both the frequencies and the total numbers of IL-2-producing CD4(+) T(E/EM) cells and of CD4(+) T(CM) cells from protected subjects were significantly higher than those from non-protected subjects. We also demonstrated for the first time that there is a strong association between the frequency of CSP peptide-reactive CD4(+) T cells producing IL-2 and the titers of CSP-specific antibodies in the same individual, suggesting that IL-2 may be acting as a growth factor for follicular Th cells and/or B cells. The frequencies of CSP peptide-reactive, TNF-α-producing CD4(+) T(E/EM) cells and of CD4(+) T(E/EM) cells secreting both IL-2 and TNF-α were also shown to be higher in protected vs. non-protected individuals. We have, therefore, demonstrated that in addition to TNF-α, IL-2 is also a significant contributing factor to RTS,S/AS vaccine induced immunity and that both T(E/EM) and T(CM) cells are major producers of IL-2.     

An open environment CT-US fusion for tissue segmentation during interventional guidance

Therapeutic ultrasound (US) can be noninvasively focused to activate drugs, ablate tumors and deliver drugs beyond the blood brain barrier. However, well-controlled guidance of US therapy requires fusion with a navigational modality, such as magnetic resonance imaging (MRI) or X-ray computed tomography (CT). Here, we developed and validated tissue characterization using a fusion between US and CT. The performance of the CT/US fusion was quantified by the calibration error, target registration error and fiducial registration error. Met-1 tumors in the fat pads of 12 female FVB mice provided a model of developing breast cancer with which to evaluate CT-based tissue segmentation. Hounsfield units (HU) within the tumor and surrounding fat pad were quantified, validated with histology and segmented for parametric analysis (fat: −300 to 0 HU, protein-rich: 1 to 300 HU, and bone: HU>300). Our open source CT/US fusion system differentiated soft tissue, bone and fat with a spatial accuracy of ∼1 mm. Region of interest (ROI) analysis of the tumor and surrounding fat pad using a 1 mm² ROI resulted in mean HU of 68±44 within the tumor and −97±52 within the fat pad adjacent to the tumor (p<0.005). The tumor area measured by CT and histology was correlated (r² = 0.92), while the area designated as fat decreased with increasing tumor size (r² = 0.51). Analysis of CT and histology images of the tumor and surrounding fat pad revealed an average percentage of fat of 65.3% vs. 75.2%, 36.5% vs. 48.4%, and 31.6% vs. 38.5% for tumors <75 mm³, 75–150 mm³ and >150 mm³, respectively. Further, CT mapped bone-soft tissue interfaces near the acoustic beam during real-time imaging. Combined CT/US is a feasible method for guiding interventions by tracking the acoustic focus within a pre-acquired CT image volume and characterizing tissues proximal to and surrounding the acoustic focus.     

PI3Ks maintain the structural integrity of T-tubules in cardiac myocytes

Background

Phosphoinositide 3-kinases (PI3Ks) regulate numerous physiological processes including some aspects of cardiac function. Although regulation of cardiac contraction by individual PI3K isoforms has been studied, little is known about the cardiac consequences of downregulating multiple PI3Ks concurrently.

Methods and Results

Genetic ablation of both p110α and p110β in cardiac myocytes throughout development or in adult mice caused heart failure and death. Ventricular myocytes from double knockout animals showed transverse tubule (T-tubule) loss and disorganization, misalignment of L-type Ca²⁺ channels in the T-tubules with ryanodine receptors in the sarcoplasmic reticulum, and reduced Ca²⁺ transients and contractility. Junctophilin-2, which is thought to tether T-tubules to the sarcoplasmic reticulum, was mislocalized in the double PI3K-null myocytes without a change in expression level.

Conclusions

PI3K p110α and p110β are required to maintain the organized network of T-tubules that is vital for efficient Ca²⁺-induced Ca²⁺ release and ventricular contraction. PI3Ks maintain T-tubule organization by regulating junctophilin-2 localization. These results could have important medical implications because several PI3K inhibitors that target both isoforms are being used to treat cancer patients in clinical trials.     

The evidence base for interventions delivered to children in primary care: an overview of cochrane systematic reviews

Background

As a first step in developing a framework to evaluate and improve the quality of care of children in primary care there is a need to identify the evidence base underpinning interventions relevant to child health. Our objective was to identify all Cochrane systematic reviews relevant to the management of childhood conditions in primary care and to assess the extent to which Cochrane reviews reflect the burden of childhood illness presenting in primary care.

Methodology/Principal Findings

We used the Cochrane Child Health Field register of child-relevant systematic reviews to complete an overview of Cochrane reviews related to the management of children in primary care. We compared the proportion of systematic reviews with the proportion of consultations in Australia, US, Dutch and UK general practice in children. We identified 396 relevant systematic reviews; 358 included primary studies on children while 251 undertook a meta-analysis. Most reviews (n = 218, 55%) focused on chronic conditions and over half (n = 216, 57%) evaluated drug interventions. Since 2000, the percentage of pediatric primary care relevant reviews only increased by 2% (7% to 9%) compared to 18% (10% to 28%) in all child relevant reviews. Almost a quarter of reviews (n = 78, 23%) were published on asthma treatments which only account for 3–5% of consultations. Conversely, 15–23% of consultations are due to skin conditions yet they represent only 7% (n = 23) of reviews.

Conclusions/Significance

Although Cochrane systematic reviews focus on clinical trials and do not provide a comprehensive picture of the evidence base underpinning the management of children in primary care, the mismatch between the focus of the published research and the focus of clinical activity is striking. Clinical trials are an important component of the evidence base and the lack of trial evidence to demonstrate intervention effectiveness in substantial areas of primary care for children should be addressed.     

Mitosis-specific phosphorylation of amyloid precursor protein at Threonine 668 leads to its altered processing and association with centrosomes

Background

Atypical expression of cell cycle regulatory proteins has been implicated in Alzheimer's disease (AD), but the molecular mechanisms by which they induce neurodegeneration are not well understood. We examined transgenic mice expressing human amyloid precursor protein (APP) and presenilin 1 (PS1) for changes in cell cycle regulatory proteins to determine whether there is a correlation between cell cycle activation and pathology development in AD.

Results

Our studies in the AD transgenic mice show significantly higher levels of cyclin E, cyclin D1, E2F1, and P-cdc2 in the cells in the vicinity of the plaques where maximum levels of Threonine 668 (Thr668)-phosphorylated APP accumulation was observed. This suggests that the cell cycle regulatory proteins might be influencing plaque pathology by affecting APP phosphorylation. Using neuroglioma cells overexpressing APP we demonstrate that phosphorylation of APP at Thr668 is mitosis-specific. Cells undergoing mitosis show altered cellular distribution and localization of P-APP at the centrosomes. Also, Thr668 phosphorylation in mitosis correlates with increased processing of APP to generate Aβ and the C-terminal fragment of APP, which is prevented by pharmacological inhibitors of the G1/S transition.

Conclusions

The data presented here suggests that cell cycle-dependent phosphorylation of APP may affect its normal cellular function. For example, association of P-APP with the centrosome may affect spindle assembly and cell cycle progression, further contributing to the development of pathology in AD. The experiments with G1/S inhibitors suggest that cell cycle inhibition may impede the development of Alzheimer's pathology by suppressing modification of βAPP, and thus may represent a novel approach to AD treatment. Finally, the cell cycle regulated phosphorylation and processing of APP into Aβ and the C-terminal fragment suggest that these proteins may have a normal function during mitosis.     

Waist Circumference,BMI, and Visceral Adipose Tissue in White Women and Women of African Descent

Although waist circumference (WC) is a marker of visceral adipose tissue (VAT), WC cut‐points are based on BMI category. We compared WC‐BMI and WC‐VAT relationships in blacks and whites. Combining data from five studies, BMI and WC were measured in 1,409 premenopausal women (148 white South Africans, 607 African‐Americans, 186 black South Africans, 445 West Africans, 23 black Africans living in United States). In three of five studies, participants had VAT measured by computerized tomography (n = 456). Compared to whites, blacks had higher BMI (29.6 ± 7.6 (mean ± s.d.) vs. 27.6 ± 6.6 kg/m², P = 0.001), similar WC (92 ± 16 vs. 90 ± 15 cm, P = 0.27) and lower VAT (64 ± 42 vs. 101 ± 59 cm², P < 0.001). The WC‐BMI relationship did not differ by race (blacks: β (s.e.) WC = 0.42 (.01), whites: β (s.e.) WC = 0.40 (0.01), P = 0.73). The WC‐VAT relationship was different in blacks and whites (blacks: β (s.e.) WC = 1.38 (0.11), whites: β (s.e.) WC = 3.18 (0.21), P < 0.001). Whites had a greater increase in VAT per unit increase in WC. WC‐BMI and WC‐VAT relationships did not differ among black populations. As WC‐BMI relationship did not differ by race, the same BMI‐based WC guidelines may be appropriate for black and white women. However, if WC is defined by VAT, race‐specific WC thresholds are required.     

Actin nemaline myopathy mouse reproduces disease, suggests other actin disease phenotypes and provides cautionary note on muscle transgene expression

Mutations in the skeletal muscle α-actin gene (ACTA1) cause congenital myopathies including nemaline myopathy, actin aggregate myopathy and rod-core disease. The majority of patients with ACTA1 mutations have severe hypotonia and do not survive beyond the age of one. A transgenic mouse model was generated expressing an autosomal dominant mutant (D286G) of ACTA1 (identified in a severe nemaline myopathy patient) fused with EGFP. Nemaline bodies were observed in multiple skeletal muscles, with serial sections showing these correlated to aggregates of the mutant skeletal muscle α-actin-EGFP. Isolated extensor digitorum longus and soleus muscles were significantly weaker than wild-type (WT) muscle at 4 weeks of age, coinciding with the peak in structural lesions. These 4 week-old mice were ~30% less active on voluntary running wheels than WT mice. The α-actin-EGFP protein clearly demonstrated that the transgene was expressed equally in all myosin heavy chain (MHC) fibre types during the early postnatal period, but subsequently became largely confined to MHCIIB fibres. Ringbinden fibres, internal nuclei and myofibrillar myopathy pathologies, not typical features in nemaline myopathy or patients with ACTA1 mutations, were frequently observed. Ringbinden were found in fast fibre predominant muscles of adult mice and were exclusively MHCIIB-positive fibres. Thus, this mouse model presents a reliable model for the investigation of the pathobiology of nemaline body formation and muscle weakness and for evaluation of potential therapeutic interventions. The occurrence of core-like regions, internal nuclei and ringbinden will allow analysis of the mechanisms underlying these lesions. The occurrence of ringbinden and features of myofibrillar myopathy in this mouse model of ACTA1 disease suggests that patients with these pathologies and no genetic explanation should be screened for ACTA1 mutations.     

KIAA0101 is overexpressed, and promotes growth and invasion in adrenal cancer

Background

KIAA0101 is a proliferating cell nuclear antigen-associated factor that is overexpressed in some human malignancies. Adrenocortical neoplasm is one of the most common human neoplasms for which the molecular causes are poorly understood. Moreover, it is difficult to distinguish between localized benign and malignant adrenocortical tumors. For these reasons, we studied the expression, function and possible mechanism of dysregulation of KIAA0101 in human adrenocortical neoplasm.

Methodology/Principal Findings

KIAA0101 mRNA and protein expression levels were determined in 112 adrenocortical tissue samples (21 normal adrenal cortex, 80 benign adrenocortical tumors, and 11 adrenocortical carcinoma (ACC). SiRNA knockdown was used to determine the functional role of KIAA0101 on cell proliferation, cell cycle, apoptosis, soft agar anchorage independent growth and invasion in the ACC cell line, NCI-H295R. In addition, we explored the mechanism of KIAA0101 dysregulation by examining the mutational status. KIAA0101 mRNA (9.7 fold) and protein expression were significantly higher in ACC (p<0.0001). KIAA0101 had sparse protein expression in only a few normal adrenal cortex samples, which was confined to adrenocortical progenitor cells. KIAA0101 expression levels were 84% accurate for distinguishing between ACC and normal and benign adrenocortical tumor samples. Knockdown of KIAA0101 gene expression significantly decreased anchorage independent growth by 80% and invasion by 60% (p = 0.001; p = 0.006). We found no mutations in KIAA0101 in ACC.

Conclusions/Significance

KIAA0101 is overexpressed in ACC. Our data supports that KIAA0101 is a marker of cellular proliferation, promotes growth and invasion, and is a good diagnostic marker for distinguishing benign from malignant adrenocortical neoplasm.     

Biophysical characteristics reveal neural stem cell differentiation potential

Background

Distinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers.

Methodology/Principal Findings

We used dielectrophoresis (DEP) to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the cells to generate neurons. DEP crossover frequency, which is a quantitative measure of cell behavior in DEP, directly correlates with neuron generation of NSPCs, indicating a potential mechanism to separate stem cells biased to particular differentiated cell fates.

Conclusions/Significance

We show here that whole cell membrane capacitance, but not membrane conductance, reflects and predicts the neurogenic potential of human and mouse NSPCs. Stem cell biophysical characteristics therefore provide a completely novel and quantitative measure of stem cell fate potential and a label-free means to identify neuron- or glial-biased progenitors.