Anti-human Vascular Endothelial Growth Factor (VEGF) Antibody Selection for Immunohistochemical Staining of Proliferating Blood Vessels

Nine commercially available vascular endothelial growth factor (VEGF) antibodies were investigated for their ability to immunostain vascular malformations (VMs) with or without immature capillary proliferation. First, all antibodies were optimized for their performance in IHC, with placenta and colon adenocarcinoma as positive control tissues. Five antibodies were regarded as unfit for VEGF immunostaining based on poor immunostaining criteria. Subsequently, Western blot analysis using VEGF rabbit polyclonal antibody (Thermo RB-9031) revealed a clear 45-kDa band in tissue extracts from VMs with immature capillary proliferation and a high Ki67-labeling index, whereas tissue extracts from mature VMs without microvascular proliferation and no Ki67-labeling index demonstrated only a very weak 45-kDa band. In contrast, two VEGF antibodies, including the popular Santa Cruz A-20, revealed bands at 45 kDa of similar intensity in tissue extracts from both types of VMs. Staining characteristics of the 45-kDa band were reflected in the results obtained in IHC. (J Histochem Cytochem 58:109–118, 2010)     

Characterization and functional analysis of the murine Frat2 gene

The Frat1 proto-oncogene was first identified as a gene contributing to tumor progression in T-cell lymphomas induced by retroviral insertional mutagenesis with the Moloney murine leukemia virus. The biological function of Frat remained elusive until its Xenopus homologue GBP was isolated as a glycogen synthase kinase 3 (GSK3)-binding protein and was shown to be an essential component of the maternal Wnt-signaling pathway. To date two Frat homologues have been described in the mouse, Frat1 and Frat3. The proteins encoded by these two genes are 84% identical. Here we describe the cloning and characterization of a third murine Frat homologue, Frat2, which is the mouse ortholog of human FRAT2. Frat1 and Frat2 are juxtaposed on chromosome 19 in a chromosomal organization conserved between man and mouse. We show that Frat1 and Frat2 are phosphorylated, which is the first evidence that these proteins are subject to posttranslational modification. Like Frat1, Frat2 is able to bind to GSK3beta. However, a side-by-side comparison of the murine Frat proteins for their capacity to induce signaling through beta-catenin/T-cell factor reveals that Frat2 is a less potent activator of the canonical Wnt pathway. Frat2 protein accumulates to higher levels upon transfection into 293T cells than either Frat1 or Frat3. Thus, whereas Frat1 may be a core component of canonical Wnt-signaling, Frat2 might very well be part of a divergent intracellular GSK3beta pathway.     

Functional expression of neurokinin 1 receptors on mast cells induced by IL-4 and stem cell factor

It is widely accepted that neurokinin 1 (NK(1)) receptors are not generally expressed on mast cells but little is known about their expression in inflammation. The present study shows expression of NK(1) receptors on bone marrow-derived mast cells (BMMC) under the influence of IL-4 or stem cell factor (SCF). Highest expression was found when both cytokines are present. Six days of coculture with the cytokines IL-4 and SCF showed significant expression of NK(1) receptors (NK(1) receptor(+)/c-kit(+) BMMC; control: 7%, IL-4/SCF: 16%), while 12 days of cytokine coculture increased this expression to 37% positive cells. A longer coculture with IL-4 and SCF did not give an additional effect. Increased expression in IL-4/SCF-treated BMMC was further confirmed using Western blot analysis. Next, we demonstrated the functional relevance of NK(1) receptor expression for mast cell activation, resulting in an enhanced degranulation upon stimulation by substance P. BMMC activation was significantly diminished by the NK(1) receptor antagonist RP67580 (10 micro M) when stimulated with low concentrations of substance P. The inactive enantiomer RP65681 had no effect. In addition, BMMC cultured from bone marrow of NK(1) receptor knockout mice showed significantly decreased exocytosis to low concentrations of substance P. The present study clearly shows that NK(1) receptor-induced activation contributes significantly at low physiological substance P concentrations (<100 micro M). In conclusion, BMMC were shown to express NK(1) receptors upon IL-4/SCF coculture. This expression of NK(1) receptors has been demonstrated to be of functional relevance and leads to an increase in the sensitivity of BMMC to substance P.     

The fellowship of the hobbit: the fauna surrounding Homo floresiensis

The Late Pleistocene Flores fauna shows a pattern observed on many other islands. It is neither aberrant nor exclusive, but the result of non‐random selective forces acting upon an impoverished and disharmonic insular fauna. By comparing the Flores vertebrate fauna with other fossil insular biotas, it is apparent that the evolution of Homo floresiensis is part of a general pattern affecting all the inhabitants of Pleistocene Flores. Vertebrate evolution on Flores appears to have been characterized by phylogenetic continuity, low species richness and a disharmonic fauna. All three aspects stem from the isolated position of the island and have resulted in the distinct morphological characteristics of the Flores fauna. Evidence reviewed herein shows that features exhibited by H. floresiensis, such as small stature, a small brain, relatively long arms, robust lower limbs and long feet, are not unique, but are shared by other insular taxa. Therefore, the evolution of H. floresiensis can be explained by existing models of insular evolution and followed evolutionary pathways similar to those of the other terrestrial vertebrates inhabiting Pleistocene Flores.     

Longitudinal Study of Performance on the Ruff Figural Fluency Test in Persons Aged 35 Years or Older

The Ruff Figural Fluency Test (RFFT) is a cognitive test to measure executive function. Longitudinal studies have shown that repeated testing improves performance on the RFFT. Such a practice effect may hinder the interpretation of test results in a clinical setting. Therefore, we investigated the longitudinal performance on the RFFT in persons aged 35–82 years. Performance on the RFFT was measured three times over an average follow-up period of six years in 2,515 participants of the Prevention of REnal and Vascular ENd-stage Disease (PREVEND) study in Groningen, the Netherlands: 53% men; mean age (SD), 53 (10) years. The effect of consecutive measurements on performance on the RFFT was investigated with linear multilevel regression models that also included age, gender, educational level and the interaction term consecutive measurement number x age as independent variables. It was found that the mean (SD) number of unique designs on the RFFT increased from 73 (26) at the first measurement to 79 (27) at the second measurement and to 83 (26) at the third measurement (p<0.001). However, the increase per consecutive measurement number was negatively associated with age and decreased with 0.23 per one-year increment of age (p<0.001). The increase per consecutive measurement number was not dependent on educational level. Similar results were found for the median (IQR) number of perseverative errors which showed a small but statistically significant increase with repeating testing: 7 (3–13) at the first measurement, 7 (4–14) at the second measurement and 8 (4–15) at the third measurement (p _trend = 0.002). In conclusion, the performance on the RFFT improved by repeating the test over an average follow-up period of three to six years. This practice effect was the largest in young adults and not dependent on educational level.     

Low levels of human immunodeficiency virus type 1 DNA in high-risk seronegative men

We detected human immunodeficiency virus type 1 (HIV-1) DNA at very low levels in sequential peripheral blood mononuclear cell samples of five out of six high-risk, seronegative, homosexual men and five out of five individuals 7.8 to 1.6 years prior to seroconversion. These data indicate a high prevalence of low-level HIV-1 DNA in exposed seronegative individuals.     

Both R5 and X4 human immunodeficiency virus type 1 variants persist during prolonged therapy with five antiretroviral drugs

A viral reservoir of human immunodeficiency virus type 1 (HIV-1)-infected, resting CD4(+) T cells persists despite suppression of plasma viremia by combination antiretroviral therapy. In a longitudinal analysis of three patients treated with a five-drug regimen, both R5 and X4 HIV-1 variants persisted in the cellular reservoir for up to 3 years.     

Exome Sequencing in Classic Hairy Cell Leukaemia Reveals Widespread Variation in Acquired Somatic Mutations between Individual Tumours Apart from the Signature BRAF V(600)E Lesion

In classic Hairy cell leukaemia (HCLc), a single case has thus far been interrogated by whole exome sequencing (WES) in a treatment naive patient, in which BRAF V(600)E was identified as an acquired somatic mutation and confirmed as occurring near-universally in this form of disease by conventional PCR-based cohort screens. It left open however the question whether other genome-wide mutations may also commonly occur at high frequency in presentation HCLc disease. To address this, we have carried out WES of 5 such typical HCLc cases, using highly purified splenic tumour cells paired with autologous T cells for germline. Apart from BRAF V(600)E, no other recurrent somatic mutation was identified in these HCLc exomes, thereby excluding additional acquired mutations as also prevalent at a near-universal frequency in this form of the disease. These data then place mutant BRAF at the centre of the neoplastic drive in HCLc. A comparison of our exome data with emerging genetic findings in HCL indicates that additional somatic mutations may however occur recurrently in smaller subsets of disease. As mutant BRAF alone is insufficient to drive malignant transformation in other histological cancers, it suggests that individual tumours utilise largely differing patterns of genetic somatic mutations to coalesce with BRAF V(600)E to drive pathogenesis of malignant HCLc disease.     

Virtual Training of the Myosignal

Objective

To investigate which of three virtual training methods produces the largest learning effects on discrete and continuous myocontrol. The secondary objective was to examine the relation between myocontrol and manual motor control tests.

Design

A cohort analytic study.

Setting

University laboratory.

Participants

3 groups of 12 able-bodied participants (N = 36).

Interventions

Participants trained the control over their myosignals on 3 consecutive days. Training was done with either myosignal feedback on a computer screen, a virtual myoelectric prosthetic hand or a computer game. Participants performed 2 myocontrol tests and 2 manual motor control tests before the first and after the last training session. They were asked to open and close a virtual prosthetic hand on 3 different velocities as a discrete myocontrol test and followed a line with their myosignals for 30 seconds as a continuous myocontrol test. The motor control tests were a pegboard and grip-force test.

Main Outcome Measures

Discrete myocontrol test: mean velocities. Continuous myocontrol test: error and error SD. Pegboard test: time to complete. Grip-force test: produced forces.

Results

No differences in learning effects on myocontrol were found for the different virtual training methods. Discrete myocontrol ability did not significantly improve as a result of training. Continuous myocontrol ability improved significantly as a result of training, both on average control and variability. All correlations between the motor control and myocontrol test outcome measures were below .50.

Conclusions

Three different virtual training methods showed comparable results when learning myocontrol. Continuous myocontrol was improved by training while discrete myocontrol was not. Myocontrol ability could not be predicted by the manual motor control tests.