Development of monoclonal antibodies specific for the human sodium-dependent phosphate co-transporter NaPi2b

Homeostasis of inorganic phosphate in the human body is maintained by regulated absorption, metabolism, and excretion. Sodium-dependent phosphate transporters (NaPi) mediate the transport of inorganic phosphate (P(i)) in cells in response to dietary phosphate consumption, hormones, and growth factors. NaPi2b is a member of the sodium-dependent phosphate transporter family, with a distinct pattern of expression and regulation. Signaling pathways activated by mitogens, glucocorticoids, and metabolic factors have been implicated in regulating P(i) transport via NaPi2b. Inactivation of NaPi2b function by mutations has been linked to human pathologies, such as pulmonary alveolar microlithiasis. In this study, we describe the generation and characterization of monoclonal antibodies against human NaPi2b. The monoclonal antibodies were shown to recognize specifically transiently overexpressed and endogenous NaPi2b in commonly used immunoassays, including Western blotting, immunoprecipitation, and immunohistochemistry. These properties make them particularly valuable reagents for elucidating NaPi2b function in health and disease.     

Examining interprofessional learning perceptions among students in a simulation-based operating room team training experience

High-fidelity simulation (HFS) operating room (OR) inter-professional team training improves healthcare students’ team-based attitudes and behaviours. Such improvements can diminish tribalism among the professions in the OR by overcoming entrenched perceptions of team members. We investigated whether simulation-based interprofessional student OR team training impacts students differently based on their professional background. From 2010 to 2013, HFS OR interprofessional student team training sessions were conducted involving senior medical students, senior undergraduate nursing students, and nurse anaesthesia students. The training involved a two-scenario format, each followed by a structured debriefing focusing on team-based competencies. Before and after each session, students completed a 15-item teamwork competencies self-efficacy survey as well as, from 2012–2013, the Readiness for Interprofessional Learning Scale (RIPLS). At the end of each session, they also completed a 6-item rating scale assessing overall team function during the session. Mean scores were calculated for each student professional group, post/pre mean differences were determined, and student t-test and ANOVA analyses were employed to compare within and between-group differences, respectively. Response rates were over 80% for each scale used. Medical students and undergraduate nursing students had significant improvements in team-based attitudes post- to pre-session. Medical students and nurse anaesthesia students had significant improvements in RIPLS scores. Statistically significant improvements from post- to pre-session were seen overall for both team-based attitudes (effect size = 0.83) and RIPLS (effect size = 0.37). The difference between the team-based scores between professions was significant; RIPLS differences were not. No significant difference existed between professions related to overall teamwork scores. HFS OR team training of healthcare students has beneficial but variable benefits for each professional group.     

Declining lymphoid progenitor fitness promotes aging-associated leukemogenesis

Aging is associated with the functional decline of cells, tissues, and organs. At the same time, age is the single most important prognostic factor in the development of most human cancers, including chronic myelogenous and acute lymphoblastic leukemias initiated by Bcr-Abl oncogenic translocations. Prevailing paradigms attribute the association between aging and cancers to the accumulation of oncogenic mutations over time, because the accrual of oncogenic events is thought to be the rate-limiting step in initiation and progression of cancers. Conversely, aging-associated functional decline caused by both cell-autonomous and non-cell-autonomous mechanisms is likely to reduce the fitness of stem and progenitor cell populations. This reduction in fitness should be conducive for increased selection of oncogenic mutations that can at least partially alleviate fitness defects, thereby promoting the initiation of cancers. We tested this hypothesis using mouse hematopoietic models. Our studies indicate that the dramatic decline in the fitness of aged B-lymphopoiesis coincides with altered receptor-associated kinase signaling. We further show that Bcr-Abl provides a much greater competitive advantage to old B-lymphoid progenitors compared with young progenitors, coinciding with restored kinase signaling pathways, and that this enhanced competitive advantage translates into increased promotion of Bcr-Abl-driven leukemias. Moreover, impairing IL-7-mediated signaling is sufficient to promote selection for Bcr-Abl-expressing B progenitors. These studies support an unappreciated causative link between aging and cancer: increased selection of oncogenic mutations as a result of age-dependent alterations of the fitness landscape.     

Role of the cofilin activity cycle in astrocytoma migration and invasion

The cofilin pathway plays a central role in the regulation of actin polymerization and the formation of cell membrane protrusions that are essential for cell migration. Overexpression of cofilin has been linked to the aggressiveness of a variety of different cancers. In these cancers, the phosphorylation of cofilin at Ser3 is a key regulatory mechanism modulating cofilin activity. The activation status of cofilin has been directly linked to tumor invasion. Accordingly, in this study, we examined the expression of cofilin and its activation status in astrocytoma cell lines and astrocytic tumors. We show that cofilin expression was increased and correlated with increasing grade malignant astrocytoma. In addition, both cofilin and LIMK had elevated expression in astrocytoma cell lines. Knockdown of cofilin by siRNA altered astrocytoma cell morphology and inhibited astrocytoma migration and invasion. Conversely, overexpression of a cofilin phosphorylation mutant in an in vivo intracranial xenograft model resulted in a more highly invasive phenotype than those xenographs expressing wild-type cofilin. Animals harboring astrocytomas stably expressing the cofilin phosphorylation mutant (cofilin-S3A) demonstrated marked local invasiveness and spread across the corpus callosum to the contralateral hemisphere in all animals. Taken together, these data indicate that the cofilin activity pathway may represent a novel therapeutic target to diminish the invasion of these highly malignant tumors.     

IL-5 expression in the sputum of patients with bronchial asthma

Expression of IL-5 mRNA and the content of IL-5 in the sputum of patients with asthma of different severity were studied before and after treatment. The expression of IL-5 mRNA in mild asthma differed from that in severe and moderate asthma before and after treatment. The level of IL-5 before therapy was different in patients with mild and severe disease. In patients with severe asthma the level of IL-5 differed before and after treatment.     

De novo mutation of GDNF, ligand for the RET/GDNFR-alpha receptor complex, in Hirschsprung disease

Hirschsprung disease (HSCR) is a common congenital abnormality characterized by absence of the enteric ganglia in the hind gut. In 10- 40% of HSCR cases, mutations of the RET receptor tyrosine kinase have been found. The recent identification of a multimeric RET ligand/receptor complex suggested that mutations of genes encoding other components of this complex might also occur in HSCR. To investigate this role, we examined the gene for glial cell line-derived neurotrophic factor (GDNF), the circulating ligand of the RET receptor complex, for mutations in a panel of sporadic and familial HSCR. We identified GDNF sequence variants in 2/36 HSCR patients. The first of these was a conservative change which did not affect the GDNF protein sequence. The second variant was a de novo missense mutation in a family with no history of HSCR and without mutation of the RET gene. Thus, our data are consistent with a causative role for GDNF mutations in some HSCR cases.      

Comparative assessment of various methods of determination of interleukin-5 production and their clinical significance in bronchial asthma

Interleukin-5 (IL-5) is an important marker of inflammation in bronchial asthma (BA). The level of IL-5 was investigated by immune-enzyme assay (IEA); the expression degree of IL-5 mRNA was studied, before and after the conducted therapy, by the inhibition reaction-IEA (IR-IEA) in sputum and blood serum of patients. No differences between contents of IL-5 were found in blood plasma of patients with various disease degrees or of patients with different BA etiologies. The IL-5 contents in sputum were reliably different in different groups and depended on a disease severity, exacerbation and remission. An evaluation of an expression degree of the IL-5 RNAm in eosinophiles, derived from patients' blood, provided for clarifying the differences between acute asthma and other disease forms and for defining the therapy influence on the parameter in question. The IL-5 RNAm expression in sputum was reliably different in patients with moderate forms and with acute forms of the disease; it was decreasing due to treatment. Finally, the results of the evaluation of the IL-5 level and the study of mRNA expression of the cytokine mutually supplement one another and make it possible to evaluate the disease degree and therapy efficiency.     

Tracking the invasiveness of human astrocytoma cells by using green fluorescent protein in an organotypical brain slice model

OBJECT: Although it is known that malignant astrocytomas infiltrate diffusely into regions of normal brain, it is frequently difficult to identify unequivocally the solitary, invading astrocytoma cell in histopathological preparations or experimental astrocytoma models. The authors describe an experimental system that facilitates the tracking of astrocytoma cells by using nonneoplastic cerebral tissue as the substrate for invasion. METHODS: Cerebral tissue was cut into 1-mm-thick slices and cultured in the upper chamber of a Transwell culture dish on top of a polyester membrane (0.4-mm pore size) that was bathed in medium supplied by the lower chamber. Two astrocytoma cell lines, U-87 MG (U87) and U343 MG-A (U343), were selected because of their differing basal cell motilities in monolayer cultures. The astrocytoma cells were stably transfected with vectors that expressed green fluorescent protein (GFP), either alone or as a fusion protein with the receptor for hyaluronic acid-mediated motility (RHAMM) in either sense or antisense orientations. Stably transfected clones that had high levels of GFP expression were selected using the direct visualization provided by fluorescence microscopy and fluorescence-activated cell-sorter analysis. The GFP-expressing astrocytoma cell clones were implanted into the center of the brain slice and the degree of astrocytoma invasion into brain tissue was measured at different time points by using the optical sectioning provided by the confocal laser microscope. The authors observed that GFP-expressing astrocytoma cells could be readily tracked and followed in this model system. Individual astrocytoma cells that exhibited green fluorescence could be readily identified following their migration through the brain slices. The GFP-labeled U87 astrocytoma cells migrated farther into the brain slice than the U343 astrocytoma cells. The RHAMM-transfected GFP-labeled astrocytoma cells also infiltrated farther than the GFP-labeled astrocytoma cells themselves. The expression of antisense RHAMM virtually abrogated the invasion of the brain slices by both astrocytoma cell lines. CONCLUSIONS: The authors believe that this organotypical culture system may be of considerable utility in studying the process of astrocytoma invasion, not only because it provides a better representation of the extracellular matrix molecules normally encountered by invading astrocytoma cells, but also because the GFP tag enables tracking of highly migratory and invasive astrocytoma cells under direct vision.