Issues in the development of medical products based on human plasma

Product development and process validation are shown in the case of several products obtained from human plasma. These are virus-inactivated plasma, intravenous immunoglobulins and the clotting factors VIII and IX. Different analytical methods are presented, which are used for product control and in-process control. For the production of virus-inactivated human plasma a down-scale protocol is presented, allowing a simulation of the production on a laboratory scale. Virus validation has shown that the reduction of transfusion-relevant viruses in the process was higher than six log steps. Determination of leachables from the RP-column, which was used in this production, proved that they appear in the final product in quantities below the detection limits only. It was also shown that the chemicals used for virus inactivation could be quantitatively removed from the product. For the isolation of other products, here intravenous gamma globulins and the clotting factors VIII and IX, similar validation steps had to be taken. In the case of clotting factor VIII the following data were determined, the reduction of viruses, the amount of leachables from the column, the residues of chemicals from the solvent/detergent treatment for virus inactivation. Virus reduction was successfully performed as well as the removal of chemicals used for virus inactivation. The amount of leachables from the columns used for chromatographic purification was found to be far below the permissible levels.     

Synthesis and Utilization of Trialkylammonium‐Substituted Cyclodextrins as Water‐Soluble Chiral NMR Solvating Agents for Anionic Compounds

Cationic trialkylammonium‐substituted α‐, β‐, and γ‐cyclodextrins containing trimethyl‐, triethyl‐, and tri‐n‐propylammonium substituent groups were synthesized and analyzed for utility as water‐soluble chiral nuclear magnetic resonance (NMR) solvating agents. Racemic and enantiomerically pure (3‐chloro‐2‐hydroxypropyl)trimethyl‐, triethyl‐, and tri‐n‐propyl ammonium chloride were synthesized from the corresponding trialkyl amine hydrochloride and either racemic or enantiomerically pure epichlorohydrin. The ammonium salts were then reacted with α‐, β‐, and γ‐cyclodextrins at basic pH to provide the corresponding randomly substituted cationic cyclodextrins. The ¹H NMR spectra of a range of anionic, aromatic compounds was recorded with the cationic cyclodextrins. Cyclodextrins with a single stereochemistry at the hydroxy group on the (2‐hydroxypropyl)trialkylammonium chloride substituent were often but not always more effective than the corresponding cyclodextrin in which the C‐2 position was racemic. In several cases, the larger triethyl or tri‐n‐propyl derivatives were more effective than the corresponding trimethyl derivative at causing enantiomeric differentiation. None of the cyclodextrin derivatives were consistently the most effective for all of the anionic compounds studied. Chirality 28:299–305, 2016. © 2016 Wiley Periodicals, Inc.     

Real-time PCR for detection and quantification,and histological characterization of <Emphasis Type="Italic">Neonectria ditissima</Emphasis> in apple trees

Key message

We designed a pair of primers from a region of the β-tubulin gene to detect and quantify Neonectria ditissima in wood of some infected apple cultivars, and optimized light microscopy to study fungal-plant interactions.

Abstract

Neone ctria ditissima, the causal pathogen of fruit tree canker, is a sordariomycete fungus that affects apple orchards, especially in north-western Europe. To prevent serious disease epidemics, an accurate, rapid, and sensitive method for detection of N. ditissima is needed for pathogen identification. A quantitative real-time PCR (qPCR) assay was developed for both detection and quantification of this pathogen in infected apple cultivars. Several primer sets were designed from regions of the β-tubulin gene. One primer set passed several validation tests, and the melting curve confirmed species-specific amplification of the correct product. In addition, the N. ditissima biomass could be detected at variable amounts in samples from the infection sites of six different cultivars, with ‘Aroma’ having the lowest amount of N. ditissima biomass and ‘Elise’ the highest. To complement the qPCR results, tissue from detached shoots and 1-year-old trees of ‘Cox’s Orange Pippin’ (susceptible) and ‘Santana’ (partially resistant) was used in a histopathology study. In both detached shoots and trees, fungal hyphae were found in cells of all tissues. No qualitative differences in the anatomy of the infected samples were observed between the cultivars. In the detached shoot experiment, both cultivars were affected but differences in the rate of disease progression suggest that the partially resistant cultivar could resist the fungus longer. The qPCR assay developed in our study produced reproducible results and can be used for detection of N. ditissima in infected trees.

     

Signalling mucin Msb2 Regulates adaptation to thermal stress in Candida albicans

Temperature is a potent inducer of fungal dimorphism. Multiple signalling pathways control the response to growth at high temperature, but the sensors that regulate these pathways are poorly defined. We show here that the signalling mucin Msb2 is a global regulator of temperature stress in the fungal pathogen Candida albicans. Msb2 was required for survival and hyphae formation at 42°C. The cytoplasmic signalling domain of Msb2 regulated temperature‐dependent activation of the CEK mitogen activated proteins kinase (MAPK) pathway. The extracellular glycosylated domain of Msb2 (100‐900 amino acid residues) had a new and unexpected role in regulating the protein kinase C (PKC) pathway. Msb2 also regulated temperature‐dependent induction of genes encoding regulators and targets of the unfolded protein response (UPR), which is a protein quality control (QC) pathway in the endoplasmic reticulum that controls protein folding/degradation in response to high temperature and other stresses. The heat shock protein and cell wall component Ssa1 was also required for hyphae formation and survival at 42°C and regulated the CEK and PKC pathways.     

Control of Adult Neurogenesis by Short-Range Morphogenic-Signaling Molecules

Adult neurogenesis is dynamically regulated by a tangled web of local signals emanating from the neural stem cell (NSC) microenvironment. Both soluble and membrane-bound niche factors have been identified as determinants of adult neurogenesis, including morphogens. Here, we review our current understanding of the role and mechanisms of short-range morphogen ligands from the Wnt, Notch, Sonic hedgehog, and bone morphogenetic protein (BMP) families in the regulation of adult neurogenesis. These morphogens are ideally suited to fine-tune stem-cell behavior, progenitor expansion, and differentiation, thereby influencing all stages of the neurogenesis process. We discuss cross talk between their signaling pathways and highlight findings of embryonic development that provide a relevant context for understanding neurogenesis in the adult brain. We also review emerging examples showing that the web of morphogens is in fact tightly linked to the regulation of neurogenesis by diverse physiologic processes.Neurogenesis in the adult mammalian brain is dynamically regulated by a number of genetic and epigenetic intrinsic factors as well as by extrinsic cues (Ninkovic and Götz 2007; Ma et al. 2010; Faigle and Song 2013). Among the latter, local signals emanating from the neural stem cell (NSC) microenvironment are thought to play a prominent modulatory role. This microenvironment, often referred to as the NSC or neurogenic “niche,” is viewed as a complex entity composed of stem and precursor cells, the surrounding mature cell types, cell-to-cell interactions, the extracellular matrix, the basal lamina, and secreted factors (Doetsch 2003). The principal mature cellular constituents of the adult NSC niches are parenchymal astroglial cells, the vasculature, microglia, and ependymal cells, all of which secrete a variety of molecules that mainly control stem-cell behavior, but also influence other stages of the adult neurogenesis process (Basak and Taylor 2009; Mu et al. 2010; Ihrie and Alvarez-Buylla 2011).As opposed to the majority of adult brain regions, the subventricular zone (SVZ) and the dentate gyrus (DG) subgranular zone (SGZ) niches are instructive milieus that allow NSC proliferation while promoting the specification and differentiation of new neurons. The relevance of the SVZ and SGZ microenvironments in adult neurogenesis was first evidenced by heterotopic transplantation experiments showing that precursor cells from a neurogenic niche, such as the SVZ, differentiate into glial cells and not into neurons when grafted to nonneurogenic areas of the brain (Seidenfaden et al. 2006). In contrast, SVZ or spinal cord precursor cells generated neurons when transplanted to a neurogenic region, such as the hippocampal DG (Suhonen et al. 1996; Shihabuddin et al. 2000). Although other in vivo studies have shown that SVZ-derived precursors maintain a certain degree of region-specific potential that is not respecified on transplantation to ectopic sites (Merkle et al. 2007), most studies suggest that local cues in the neurogenic brain niches are key for neuronal differentiation to occur. On the other hand, combined transplantation of both NSCs and niche cells to nonneurogenic areas, or expression of niche factors at the site where NSCs are grafted, promotes neuronal differentiation (Lim et al. 2000, Jiao and Chen 2008). Thus, it has progressively become apparent that extrinsic signals produced by niche cells enable the adult neurogenic program to proceed.More recently, transgenic and virus-based approaches allowing cell type- and temporal-specific manipulation of gene expression in the niches have provided great insights into the identity of the extrinsic signals regulating neurogenesis in vivo and into the molecular mechanisms elicited by those signals. Several soluble and membrane-bound factors have been identified as determinants of SVZ and SGZ neurogenesis, including morphogens, growth factors, neurotrophins, and neurotransmitters. Among these determinants, morphogens are ideally suited to fine-tune the sophisticated processes of stem-cell activation, progenitor expansion, and differentiation required for proper adult neurogenesis. Morphogens are defined as signaling molecules that pattern developing tissues in a concentration-dependent manner (Ashe and Briscoe 2006; Rogers and Schier 2011). They mostly operate in long-range gradients created by synthesis and diffusion of the morphogen proteins from a source and clearance during their flux by diverse mechanisms, such as immobilization, degradation, or endocytosis. Additional molecules that act as anti- or promorphogens further refine their activity. It is important to note that, although morphogens are graded signals, the response they elicit is not graded. Small differences in the concentration of a morphogen can trigger sharp thresholds in the expression of target genes. In addition, morphogens can also act at short range. Lipidation and low-affinity interactions with extracellular matrix components confine the movement of some morphogen proteins and promote effective morphogen–receptor interactions at the cell surface. Cells exposed locally to different morphogen doses respond by adopting different fates and, in this way, a morphogen can assign positional information to cells within a structure or territory, such as a stem-cell niche, and provoke different niche responses or outputs depending on the context (Ashe and Briscoe 2006; Rogers and Schier 2011).Here, we review our current understanding of the role and mechanisms of short-range niche morphogens, including ligands from the Wnt, Notch, Sonic hedgehog, and bone morphogenetic protein (BMP) families, in the regulation of adult neurogenesis. We discuss cross talk between their signaling pathways and intersection with other signaling pathways operating in the niches. We also highlight findings and emerging principles of embryonic development that provide a relevant context for understanding the growing field of adult neurogenesis.     

Enterovirus D-68 Infection,Prophylaxis, and Vaccination in a Novel Permissive Animal Model,the Cotton Rat (Sigmodon hispidus)

In recent years, there has been a significant increase in detection of Enterovirus D-68 (EV-D68) among patients with severe respiratory infections worldwide. EV-D68 is now recognized as a re-emerging pathogen; however, due to lack of a permissive animal model for EV-D68, a comprehensive understanding of the pathogenesis and immune response against EV-D68 has been hampered. Recently, it was shown that EV-D68 has a strong affinity for α2,6-linked sialic acids (SAs) and we have shown previously that α2,6-linked SAs are abundantly present in the respiratory tract of cotton rats (Sigmodon hispidus). Thus, we hypothesized that cotton rats could be a potential model for EV-D68 infection. Here, we evaluated the ability of two recently isolated EV-D68 strains (VANBT/1 and MO/14/49), along with the historical prototype Fermon strain (ATCC), to infect cotton rats. We found that cotton rats are permissive to EV-D68 infection without virus adaptation. The different strains of EV-D68 showed variable infection profiles and the ability to produce neutralizing antibody (NA) upon intranasal infection or intramuscular immunization. Infection with the VANBT/1 resulted in significant induction of pulmonary cytokine gene expression and lung pathology. Intramuscular immunization with live VANBT/1 or MO/14/49 induced strong homologous antibody responses, but a moderate heterologous NA response. We showed that passive prophylactic administration of serum with high content of NA against VANBT/1 resulted in an efficient antiviral therapy. VANBT/1-immunized animals showed complete protection from VANBT/1 challenge, but induced strong pulmonary Th1 and Th2 cytokine responses and enhanced lung pathology, indicating the generation of exacerbated immune response by immunization. In conclusion, our data illustrate that the cotton rat is a powerful animal model that provides an experimental platform to investigate pathogenesis, immune response, anti-viral therapies and vaccines against EV-D68 infection.     

Myosin isoform transitions in regeneration of fast and slow muscles during postnatal development of the rat

Regeneration of rat fast (gastrocnemius medialis) and slow (soleus) muscles was examined after degeneration of myofibers had been achieved by injection of cardiotoxin into the hindleg during the first week after birth. Myogenesis in the regenerating muscles was compared to postnatal myogenesis in the contralateral and in control muscles. Synthesis of embryonic and neonatal myosin isoforms was initiated 3 days after injury. These forms were gradually replaced by the intermediate and fast adult isoforms (type II fiber myosins), whose synthesis followed the same curve in regenerating, contralateral, and control muscles. In contrast, synthesis of the slow myosin isoform (type I fiber myosin) was greatly delayed in injured muscles, but eventually became equal to its synthesis in contralateral and control muscles. It therefore appears that synthesis of type II fiber myosins is similarly regulated, probably by thyroid hormone, in developing regenerating and normal muscles, while synthesis of type I fiber myosin depends on other factor(s).