Imaging Flow Cytometry for Sizing and Counting of Subvisible Particles in Biotherapeutics.

Imaging flow cytometry (IFC), a technique originally designed for cellular imaging, is featured by the parallel acquisition in brightfield (BF), fluorescence (FL), and side scattering channels. Introduced to the field of subvisible particle analysis in biopharmaceuticals roughly ten years ago, it has the potential to yield additional information, e.g., on particle origin. Here, we present an extensive, systematic development of masks for IFC image analysis to optimize the accuracy of size determination of polystyrene beads and pharmaceutically relevant particles (protein, silicone oil) in BF and FL channels. Based on the developed masks, particle sizing and counting by IFC are compared to flow imaging microscopy (FIM). Mask verification based on fluorescent polystyrene particles revealed good agreement between sizes obtained from IFC and FIM. In the evaluation of counting accuracy, IFC reported lower concentrations for polystyrene particle standards than FIM. For the analysis of fluorescently stained silicone oil and protein particles however, IFC FL imaging reported higher particle concentrations in the low micrometer size range. Overall, we identified IFC as suitable tool to generate supportive data for particle characterization purposes or trouble shooting activities, but not as routine quantitative technique, e.g., for subvisible particle analysis during drug product development or quality control.

Specific subtypes of cutaneous mechanoreceptors require neurotrophin-3 following peripheral target innervation.

Neurotrophin-3 (NT-3) is required for the development of most sensory neurons of the dorsal root ganglia. Using electrophysiological techniques in mice with null mutations of the NT-3 gene, we show that two functionally specific subsets of cutaneous afferents differentially require this factor: D-hair receptors and slowly adapting mechanoreceptors; other cutaneous receptors were unaffected. Merkel cells, which are the end organs of slowly adapting mechanoreceptors, are virtually absent in 14-day-old homozygous mutants and are severely reduced in adult NT-3 heterozygous animals. This loss of Merkel cells, together with their innervation, happens in the first postnatal weeks of life, in contrast to muscle spindles and afferents, which are never formed in the absence of NT-3. Thus, NT-3 is essential for the maintenance of specific cutaneous afferents known to subserve fine tactile discrimination in humans.

Trigger-happy resident memory CD4+ T cells inhabit the human lungs.

Resident memory T cells (TRM) reside in the lung epithelium and mediate protective immunity against respiratory pathogens. Although lung CD8+ TRM have been extensively characterized, the properties of CD4+ TRM remain unclear. Here we determined the transcriptional signature of CD4+ TRM, identified by the expression of CD103, retrieved from human lung resection material. Various tissue homing molecules were specifically upregulated on CD4+ TRM, whereas expression of tissue egress and lymph node homing molecules were low. CD103+ TRM expressed low levels of T-bet, only a small portion expressed Eomesodermin (Eomes), and although the mRNA levels for Hobit were increased, protein expression was absent. On the other hand, the CD103+ TRM showed a Notch signature. CD4+CD103+ TRM constitutively expressed high transcript levels of numerous cytotoxic mediators that was functionally reflected by a fast recall response, magnitude of cytokine production, and a high degree of polyfunctionality. Interestingly, the superior cytokine production appears to be because of an accessible interferon-γ (IFNγ) locus and was partially because of rapid translation of preformed mRNA. Our studies provide a molecular understanding of the maintenance and potential function of CD4+ TRM in the human lung. Understanding the specific properties of CD4+ TRM is required to rationally improve vaccine design.

Lateralized effects on reaching by children.

This study examined the limb selection profiles of children for a reach-to-grasp task presented in various positions of hemispace. Underlying questions focused on the use of attentional information and lateralized effects in motor programming for reaching movements. As expected, both right- and left-handed groups used their dominant limb more frequently at the midline and in their own ipsilateral hemispace. However, in response to stimuli presented in contralateral (to the dominant limb) hemispace, both groups switched to using their nondominant limbs at significant levels. As a general comparison, right-handers exhibited greater use of their dominant limb, but arguably, motor dominance in this context may have intervened with the participant's ability to use attentional information to produce a more efficient response. Overall, these findings address the phenomenon associated with motor dominance and use of attentional information in programming.

[Time-resolved MR angiography of the renal artery: morphology and perfusion]. / Zeitaufgelöste MR-Angiographie der Nierenarterien: Morphologie und Perfusion.

PURPOSE: To prove the hypothesis that renal artery stenosis and changes in renal perfusion can be detected with contrast-enhanced time-resolved MR angiography in a single examination. MATERIAL AND METHODS: In 71 patients, 137 renal arteries and 14 accessory renal arteries were studied. The examinations were performed on a 1.5 T system. A T 1 -weighted gradient echo sequence with a temporal resolution of 7 s was used. Single dose of contrast material (0.1 mmol/kg Gd-DTPA) was injected with a power injector with a flow rate of 2 ml/s. Criterion for the assessment of renal perfusion was the slope ratio of the signal intensity time curve in both kidneys. RESULTS: Forty renal artery stenoses and one occlusion of a renal artery were detected. In 48 kidneys (35 %) segmental arteries were evaluated. The accuracy of the slope ratio (limit value 0.75) concerning the detection of unilateral renal artery stenosis was 92.6 % (sensitivity 75 %, specificity 95.7 %). CONCLUSION: Time-resolved MR angiography can detect changes in renal perfusion in patients with unilateral renal artery stenosis.

Induction of cell proliferation by fibroblast and insulin-like growth factors in pure rat inner ear epithelial cell cultures.

Proliferation of supporting cells in the inner ear is the early major event occurring during hair cell regeneration after acoustic trauma or aminoglycoside treatment. In the present study, we examined the possible influence of 30 growth factors on the proliferation of pure rat utricular epithelial cells in culture. Utricular epithelial sheets were separated and partially dissociated from early postnatal rats via a combined enzymatic and mechanical method. The cultured utricular epithelial cells expressed exclusively epithelial cell antigens, but not fibroblast, glial, or neuronal antigens. With tritiated thymidine incorporation assays, we found that several fibroblast growth factor (FGF) family members, insulin-like growth factor-1 (IGF-1), IGF-2, transforming growth factor-alpha (TGF-alpha), and epidermal growth factor (EGF), stimulated proliferation of the utricular epithelial cells. In contrast, neurotrophins and other growth factors did not elicit any detectable mitogenic effects. Among all of the growth factors examined, FGF-2 was the most potent mitogen. When FGF-2 was added in combination with IGF-1 or TGF-alpha to the medium, combined effects were seen. These results were confirmed with BrdU immunocytochemistry. Thus, the present culture system provides a rapid and reliable assay system to screen novel growth factors involved in proliferation of mammalian inner ear supporting cells. Furthermore, immunostainings revealed that the cultured utricular epithelial cells expressed FGF and IGF-1 receptors, and utricular hair cells produced FGF-2 in vivo. The addition of neutralizing antibodies against FGF-2 or IGF-1 to the cultures significantly inhibited the utricular epithelial cell proliferation. This work suggests that FGF-2 and IGF-1 may regulate the proliferation step during hair cell development and regeneration.