Challenges with Developing In Vitro Dissolution Tests for Orally Inhaled Products (OIPs)

The purpose of this article is to review the suitability of the analytical and statistical techniques that have thus far been developed to assess the dissolution behavior of particles in the respirable aerodynamic size range, as generated by orally inhaled products (OIPs) such as metered-dose inhalers and dry powder inhalers. The review encompasses all analytical techniques publicized to date, namely, those using paddle-over-disk USP 2 dissolution apparatus, flow-through cell dissolution apparatus, and diffusion cell apparatus. The available techniques may have research value for both industry and academia, especially when developing modified-release formulations. The choice of a method should be guided by the question(s) that the research strives to answer, as well as by the strengths and weaknesses of the available techniques. There is still insufficient knowledge, however, for translating the dissolution data into statements about quality, performance, safety, or efficacy of OIPs in general. Any attempts to standardize a dissolution method for compendial inclusion or compendial use would therefore be premature. This review reinforces and expands on the 2008 stimulus article of the USP Inhalation Ad Hoc Advisory Panel, which "could not find compelling evidence suggesting that such dissolution testing is kinetically and/or clinically crucial for currently approved inhalation drug products."     

Non-tuberculous mycobacteria and their surface lipids efficiently induced IL-17 production in human T cells

Interleukin-17 (IL-17) is produced by a subset of CD4⁺ T helper (Th) lymphocytes known as Th17 cells. In humans, IL-1β, enhanced by IL-6 and IL-23 is crucial for differentiation of these cells. IL-17 evokes inflammation and is involved in host defence against microorganisms, although little is known about its role in diseases caused by non-tuberculous mycobacteria. The genus Mycobacterium contains both obligate and opportunistic pathogens as well as saprophytes, and the mycobacterial cell envelope is unique in its abundance of lipids. Here we investigated IL-17 and IL-23 production in human PBMC in response to intact UV-inactivated mycobacteria and mycobacterial surface lipids from two opportunistic (Mycobacterium avium and Mycobacterium abscessus) and one generally non-pathogenic (Mycobacterium gordonae) species. Representative Gram-positive (Enterococcus faecalis, Streptococcus mitis) and Gram-negative (Escherichia coli) bacteria were included as controls. Intact mycobacteria induced production of large amounts of IL-17, while IL-17 responses to control bacteria were negligible. Purified CD4⁺ T cells, but not CD4-depleted cell fractions, produced this IL-17. Isolated mycobacterial surface lipids induced IL-17, but not IL-23 production. The ability of the non-tuberculous mycobacteria to induce IL-17 production in CD4⁺ T cells was the same regardless of the pathogenic potential of the particular mycobacterial species.     

The surface lipids of non-tuberculous mycobacteria suppress production of phagocyte activating cytokines in human peripheral blood mononuclear cells

The genus Mycobacterium includes obligate pathogens as well as opportunistic and non-pathogenic species ubiquitous in the environment. Mycobacteria have a unique cell wall abundant in lipids. Here we investigated cytokine production by human peripheral blood mononuclear cells (PBMC) in response to the opportunistic mycobacteria Mycobacterium avium and Mycobacterium abscessus, the non-pathogenic Mycobacterium gordonae and extracted surface lipids from the three species. The cytokine response elicited by mycobacteria, regardless of their pathogenic potential, differed distinctly from that induced by control Gram-positive (Enterococcus faecalis, Streptococcus mitis) and Gram-negative (Escherichia coli) bacteria. Mycobacteria induced no IL-12 and less TNF and IFN-γ compared with conventional Gram-positive bacteria. IL-10 was induced by all the mycobacteria and this production was partly responsible for the down-regulation of IL-12 and IFN-γ. The capacity of the Gram-positive bacterium E. faecalis to induce IL-12, as well as TNF and IFN-γ, in human PBMCs was strongly reduced when mycobacterial lipids were added. The mycobacterial surface lipids down-regulated the production of IL-12 and IFN-γ without eliciting IL-10 production. Our results show that mycobacteria evade triggering production of phagocyte activating cytokines (IL-12, TNF and IFN-γ) and that the mycobacterial cell wall surface lipids may play a significant role in this process.     

Functional metagenomics unveils a multifunctional glycosyl hydrolase from the family 43 catalysing the breakdown of plant polymers in the calf rumen

Microbial communities from cow rumen are known for their ability to degrade diverse plant polymers at high rates. In this work, we identified 15 hydrolases through an activity-centred metagenome analysis of a fibre-adherent microbial community from dairy cow rumen. Among them, 7 glycosyl hydrolases (GHs) and 1 feruloyl esterase were successfully cloned, expressed, purified and characterised. The most striking result was a protein of GH family 43 (GHF43), hereinafter designated as R_09-02, which had characteristics very distinct from the other proteins in this family with mono-functional β-xylosidase, α-xylanase, α-L-arabinase and α-L-arabinofuranosidase activities. R_09-02 is the first multifunctional enzyme to exhibit β-1,4 xylosidase, α-1,5 arabinofur(pyr)anosidase, β-1,4 lactase, α-1,6 raffinase, α-1,6 stachyase, β-galactosidase and α-1,4 glucosidase activities. The R_09-02 protein appears to originate from the chromosome of a member of Clostridia, a class of phylum Firmicutes, members of which are highly abundant in ruminal environment. The evolution of R_09-02 is suggested to be driven from the xylose- and arabinose-specific activities, typical for GHF43 members, toward a broader specificity to the glucose- and galactose-containing components of lignocellulose. The apparent capability of enzymes from the GHF43 family to utilise xylose-, arabinose-, glucose- and galactose-containing oligosaccharides has thus far been neglected by, or could not be predicted from, genome and metagenome sequencing data analyses. Taking into account the abundance of GHF43-encoding gene sequences in the rumen (up to 7% of all GH-genes) and the multifunctional phenotype herein described, our findings suggest that the ecological role of this GH family in the digestion of ligno-cellulosic matter should be significantly reconsidered.     

The S. pombe cytokinesis NDR kinase Sid2 activates Fin1 NIMA kinase to control mitotic commitment through Pom1/Wee1

Mitotic exit integrates the reversal of the phosphorylation events initiated by mitotic kinases with a controlled cytokinesis event that cleaves the cell in two. The mitotic exit network (MEN) of budding yeast regulates both processes, whereas the fission yeast equivalent, the septum initiation network (SIN), controls only the execution of cytokinesis. The components and architecture of the SIN and MEN are highly conserved. At present, it is assumed that the functions of the core SIN-MEN components are restricted to their characterized roles at the end of mitosis. We now show that the NDR (nuclear Dbf2-related) kinase component of the fission yeast SIN, Sid2-Mob1, acts independently of the other known SIN components in G2 phase of the cell cycle to control the timing of mitotic commitment. Sid2-Mob1 promotes mitotic commitment by directly activating the NIMA (Never In Mitosis)-related kinase Fin1. Fin1's activation promotes its own destruction, thereby making Fin1 activation a transient feature of G2 phase. This spike of Fin1 activation modulates the activity of the Pom1/Cdr1/Cdr2 geometry network towards?Wee1.     

PAI-1-dependent endothelial cell death determines severity of radiation-induced intestinal injury

Normal tissue toxicity still remains a dose-limiting factor in clinical radiation therapy. Recently, plasminogen activator inhibitor type 1 (SERPINE1/PAI-1) was reported as an essential mediator of late radiation-induced intestinal injury. However, it is not clear whether PAI-1 plays a role in acute radiation-induced intestinal damage and we hypothesized that PAI-1 may play a role in the endothelium radiosensitivity. In vivo, in a model of radiation enteropathy in PAI-1 -/- mice, apoptosis of radiosensitive compartments, epithelial and microvascular endothelium was quantified. In vitro, the role of PAI-1 in the radiation-induced endothelial cells (ECs) death was investigated. The level of apoptotic ECs is lower in PAI-1 -/- compared with Wt mice after irradiation. This is associated with a conserved microvascular density and consequently with a better mucosal integrity in PAI-1 -/- mice. In vitro, irradiation rapidly stimulates PAI-1 expression in ECs and radiation sensitivity is increased in ECs that stably overexpress PAI-1, whereas PAI-1 knockdown increases EC survival after irradiation. Moreover, ECs prepared from PAI-1 -/- mice are more resistant to radiation-induced cell death than Wt ECs and this is associated with activation of the Akt pathway. This study demonstrates that PAI-1 plays a key role in radiation-induced EC death in the intestine and suggests that this contributes strongly to the progression of radiation-induced intestinal injury.     

Plasma membrane calcium pump (PMCA) isoform 4 is targeted to the apical membrane by the w-splice insert from PMCA2

Local Ca(2+) signaling requires proper targeting of the Ca(2+) signaling toolkit to specific cellular locales. Different isoforms of the plasma membrane Ca(2+) pump (PMCA) are responsible for Ca(2+) extrusion at the apical and basolateral membrane of polarized epithelial cells, but the mechanisms and signals for differential targeting of the PMCAs are not well understood. Recent work demonstrated that the alternatively spliced w-insert in PMCA2 directs this pump to the apical membrane. We now show that inserting the w-insert into the corresponding location of the PMCA4 isoform confers apical targeting to this normally basolateral pump. Mutation of a di-leucine motif in the C-tail thought to be important for basolateral targeting did not enhance apical localization of the chimeric PMCA4(2w)/b. In contrast, replacing the C-terminal Val residue by Leu to optimize the PDZ ligand site for interaction with the scaffolding protein NHERF2 enhanced the apical localization of PMCA4(2w)/b, but not of PMCA4x/b. Functional studies showed that both apical PMCA4(2w)/b and basolateral PMCA4x/b handled ATP-induced Ca(2+) signals with similar kinetics, suggesting that isoform-specific functional characteristics are retained irrespective of membrane targeting. Our results demonstrate that the alternatively spliced w-insert provides autonomous apical targeting information in the PMCA without altering its functional characteristics.     

Human neurocysticercosis: in vivo expansion of peripheral regulatory T cells and their recruitment in the central nervous system

Human neurocysticercosis (NC) is caused by Taenia solium larvae lodged in the central nervous system. Most cases occur with no, or mild, neurological symptoms. However, in some patients, neuroinflammation is exacerbated, leading to severe forms of the disease. Considering the critical role of regulatory T cells (Tregs) in balancing inflammation in chronic diseases, their participation in restraining the inflammatory response in NC was explored in the present study. The frequency of Tregs and their relationship with the level of the proliferative response, the level of activated lymphocytes, and the cytokines expressed were determined in severe NC patients compared with those from healthy donors. Significantly increased peripheral Tregs (CD4(+)CD25(high) and CD4(+)CD25(high)FoxP3(+), CD4(+)CD25(high)CTLA4(+), and CD4(+)CD25(high) IL10(+)) and a significant decrease in activated (CD38(+) and CD69(+)) T cells were observed in 19 NC patients versus 10 healthy subjects. Significantly increased Tregs in NC are accompanied by a depressed specific, and non-specific, lymphocyte proliferative response, and they negatively correlate with activated CD4(+)CD69(+) lymphocytes. Treg frequencies were also determined in cerebral spinal fluid for 8 of the 19 NC patients. A positive significant correlation between peripheral and local Tregs was observed. Here, we report for the first time data that support the possible contribution of local and systemic Tregs in limiting neuroinflammation in NC.     

Extracellular signal-regulated kinase 1/2 signaling promotes oligodendrocyte myelination in vitro

J. Neurochem. (2012) 122, 1167-1180. ABSTRACT: Multiple extracellular factors have been implicated in orchestrating myelination of the CNS; however, less is known about the intracellular signaling cascades that regulate this process. We have previously shown that brain-derived neurotrophic factor (BDNF) promotes oligodendrocyte myelination. Here, we screened for the activation of candidate signaling pathways in in vitro myelination assays and found that extracellular signal-regulated kinase (Erk) signaling positively correlated with basal levels of oligodendrocyte myelination as well as BDNF-induced myelination in vitro. By selectively manipulating Erk1/2 activation in oligodendrocytes in vitro, we found that constitutive activation of Erk1/2 significantly increased myelination, mimicking the promyelinating effect of BDNF, and also caused myelination to occur earlier. Conversely, selective inhibition of Erk1/2 in oligodendrocytes significantly reduced the basal level of myelination and blocked the promyelinating effect of BDNF. Analysis of myelinating spinal cord and corpus callosum white matter tracts revealed that the majority of mature oligodendrocytes are co-labeled with phospho-Erk1/2, whereas phospho-Erk1/2 was rarely observed in oligodendrocyte progenitor cells. Finally, the total level of phospho-Erk1/2 correlated with myelin formation during the early postnatal period. Collectively, these data identify that Erk1/2 signaling within oligodendrocytes exerts an important and direct effect to promote myelination.     

Infant B cell memory differentiation and early gut bacterial colonization

Germ-free animal models have demonstrated that commensal bacterial colonization of the intestine induces B cell differentiation and activation. Whether colonization with particular bacterial species or groups is associated with B cell development during early childhood is not known. In a prospective newborn/infant cohort including 65 Swedish children, we examined the numbers and proportions of CD20(+), CD5(+), and CD27(+) B cells in blood samples obtained at several time points during the first 3 y of life using flow cytometry. Fecal samples were collected and cultured quantitatively for major facultative and anaerobic bacteria at 1, 2, 4, and 8 wk of life. We found that the numbers of CD20(+) B cells and CD5(+)CD20(+) B cells reached their highest levels at 4 mo, whereas CD20(+) B cells expressing the memory marker CD27 were most numerous at 18 and 36 mo of age. Using multivariate analysis, we show that early colonization with Escherichia coli and bifidobacteria were associated with higher numbers of CD20(+) B cells that expressed the memory marker CD27 at 4 and 18 mo of age. In contrast, we were unable to demonstrate any relation between bacterial colonization pattern and numbers of CD20(+) or CD5(+)CD20(+) B cells. These results suggest that the intestinal bacterial colonization pattern may affect the B cell maturation also in humans, and that an early gut microbiota including E. coli and bifidobacteria might promote this maturation.