Rat primary hepatocytes show enhanced performance and sensitivity to acetaminophen during three-dimensional culture on a polystyrene scaffold designed for routine use

The in vitro evaluation of hepatotoxicity is an essential stage in the research and development of new pharmaceuticals as the liver is one of the most commonly impacted organs during preclinical toxicity studies. Fresh primary hepatocytes in monolayer culture are the most commonly used in vitro model of the liver but often exhibit limited viability and/or reduction or loss of important liver-specific functions. These limitations could potentially be overcome using three-dimensional (3D) culture systems, but their experimental nature and limited use in liver toxicity screening and drug metabolism has impaired their uptake into commercial screening programs. In this study we use a commercially available polystyrene scaffold developed for routine 3D cell culture to maintain primary rat hepatocytes for use in metabolism and toxicity studies over 72?h. We show that primary hepatocytes retain their natural cuboidal morphology with significantly higher viability (>74%) than cells grown in monolayer culture (maximum of 57%). Hepatocytes in the 3D scaffolds exhibit differential expression of genes associated with phase I, II, and III drug metabolism under basal conditions compared with monolayer culture and can be induced to stably express significantly higher levels of the cytochrome-P450 enzymes 1A2, 2B1, and 3A2 over 48?h. In toxicity studies the hepatocytes in the 3D scaffolds also show increased sensitivity to the model toxicant acetaminophen. These improvements over monolayer culture and the availability of this new easy to use 3D scaffold system could facilitate the uptake of 3D technologies into routine drug screening programs.     

Identification of polyphenolic compounds and black tea extract as potent inhibitors of lipid membrane destabilization by Aβ₄₂ aggregates

Amyloid-β (Aβ) aggregation is a recognized key process in the pathogenesis of Alzheimer's disease (AD). Misfolded Aβ peptides self-assemble into higher-order oligomers that compromise membrane integrity, leading to synaptic degeneration and neuronal cell death. The main aim of this study was to explore whether small-molecule compounds and black tea extract can protect phospholipid membranes from disruption by Aβ aggregates. We first established a robust protocol for aggregating Aβ?? peptides into a range of oligomers that efficiently permeabilized small unilamellar liposomes. Next, 15 natural plant polyphenolic compounds, 8 N'-benzylidene-benzohydrazide (NBB) compounds and black tea extract were assessed for their ability to antagonize liposome permeabilization by the Aβ?? oligomers. Our data indicates that black tea extract, the flavones apigenin and baicalein, and the stilbene nordihydroguaiaretic acid (NDGA) are indeed potent inhibitors. Taking into consideration the results of all the small-molecule polyphenols and NBB compounds, it can be proposed that a dihydroxyphenyl ring structure, alone or as part of a flavone scaffold, is particularly effective for protection against membrane damage by the Aβ?? oligomers. Given the critical role of membrane perforation in the neurodegenerative cascade, these conclusions may guide the design and development of novel therapeutic drugs in AD.     

A gene family expressing a host-protective antigen of Echinococcus granulosus.

Echinococcus granulosus causes cystic hydatidosis in humans. A recombinant antigen vaccine has been developed, for use in the parasite's natural animal intermediate hosts, that may provide a new tool for control of hydatid disease transmission. The antigen, designated EG95, is encoded by a cDNA the features of which indicate it to be an incomplete copy of the associated mRNA. Characterisation of the gene(s) encoding the antigen was undertaken in order to enable subsequent study of genetic variability in the gene and associated protein in different parasite isolates. Southern hybridisation studies of E. granulosus genomic DNA probed with the eg95 cDNA revealed that the gene belonged to a gene family. DNA sequence analysis of cloned genomic fragments indicated that the gene family consists of at least seven members, one of which is a pseudogene. The gene having identity with the eg95 cDNA was cloned and sequenced, and the full length mRNA characterised. Genomic sequence and structure of the eg95 gene family members are highly conserved with respect to the gene encoding EG95. Four eg95-related genes are predicted to express an identical EG95 protein and all four were shown to be expressed in the oncosphere life-cycle stage. The full length EG95 protein has a predicted molecular mass of 16.9 kDa, secretory signal sequence, carboxy-terminal glycosylphosphatidylinositol hydrophobic anchor motif and a fibronectin type III domain. PCR amplification conditions were established which allow gene-specific characterisation of the eg95 gene in E. granulosus isolates from different host species and geographical locations.     

The efficacy of intranasal interferon alpha-2a in respiratory syncytial virus infection in volunteers

In a double-blind, placebo-controlled study, self-administered intranasal interferon alpha-2a or placebo was given both before and after challenge with respiratory syncytial virus. The incidence of colds and the severity of signs and symptoms were reduced in those receiving interferon alpha-2a as compared with those given placebo. In a further double-blind, placebo-controlled study, self-administered interferon alpha-2a or placebo was given only to those volunteers who developed colds following challenge with respiratory syncytial virus. There was no evidence that interferon alpha-2a reduced the severity of the signs and symptoms or shortened the duration of the illness. The similarity of these results to the effect of interferon alpha-2a in rhinovirus infections in volunteers is discussed.     

Organ culture of human foetal pancreas: conditions which affect basal and stimulated insulin release

Human foetal pancreas has been maintained in organ culture with net synthesis and release of insulin for up to 60 days. The age of the donor foetus affected the basal insulin release rate. A plateau of secretion was reached with foetuses of greater than or equal to 16 weeks of gestation. Explants cultured within 2 h of expulsion following prostaglandin induced termination secreted 3.0 times more insulin after 20 days of culture than those cultured within 2-4 h and 8.1 times more than those cultured more than 4 h post-termination. A high oxygen environment was toxic to the explants during culture. Fresh tissue responded to a high concentration of glucose (19.3 mM) with a small but significant increase in insulin secretion. The addition of 10 mM theophylline caused a major increase in insulin release. Cultured tissue did not respond to glucose alone but did not show increased insulin release following stimulation with glucose (22 mM) together with theophylline (10 mM) in static incubation. The culture of human foetal pancreatic tissue may be useful in maintaining responsive beta cells and may help to ensure an adequate amount of donor tissue for future transplantation into diabetic patients.     

Detection in allergic individuals of IgE specific for the Australian paralysis tick, Ixodes holocyclus

Three techniques were developed to detect IgE specific to Ixodes holocyclus. The radioimmunoassay (RIA) was found to be superior to both radioallergosorbent test and enzyme immunoassay techniques. Validation of the RIA technique was provided by dilution experiments and by a blind study involving 13 volunteers; the RIA graph was linear over a wide range of concentrations. The RIA ratio clearly discriminated between those volunteers with and without systemic allergic reactions and helped to identify borderline cases showing local or cutaneous allergic reactions. It appears to be suitable for diagnostic and research applications.     

HOXB13 is a sensitive and specific marker of prostate cells, useful in distinguishing between carcinomas of prostatic and urothelial origin

The origin of a primary or metastatic carcinoma in the pelvic area is sometimes difficult to establish, in particular the distinction between those originating in the bladder and the prostate. A candidate marker is the HOXB13 gene, essential for prostate development. Some studies have shown expression of HOXB13 protein by immunohistochemistry in the nuclear compartment of benign prostate luminal epithelium and prostate carcinoma. Forty-two cases of biopsies and resection specimens of the prostate and urinary bladder, metastatic lymph nodes, and pelvic masses were retrieved from our databases. In all cases, doubt persisted regarding prostatic versus urothelial origin. All cases were stained for CK7, p63, p504s, PSA, CK20, and HOXB13. Chromogranin A, CD56, and synaptophysin were used when neuroendocrine differentiation was suspected. HOXB13 staining was negative or only weakly positive in all carcinomas of urothelial origin. Three of four carcinomas with neuroendocrine differentiation did not express HOXB13. The fourth carcinoma, in a patient with a history of prostate carcinoma, was positive. In two cases with a synchronous prostatic and urothelial carcinoma, HOXB13 was exclusively expressed in the prostatic carcinoma. Our results demonstrate that HOXB13 expression identifies prostatic origin of a carcinoma with good sensitivity (89 %) and very good specificity (100 %). HOXB13 is a specific and sensitive marker for prostate cells and a valuable diagnostic tool, especially when poorly differentiated or neuroendocrine tumors are encountered. These results justify testing of HOXB13 as a prostate-specific carcinoma marker in larger cohorts for a more thorough evaluation of its sensitivity and specificity.     

External rib structure can be predicted using mathematical models: An anatomical study with application to understanding fractures and intercostal muscle function

As ribs adapt to stress like all bones, and the chest behaves as a pressure vessel, the effect of stress on the ribs can be determined by measuring rib height and thickness. Rib height and thickness (depth) were measured using CT scans of seven rib cages from anonymized cadavers. A Finite Element Analysis (FEA) model of a rib cage was constructed using a validated approach and used to calculate intramuscular forces as the vectors of both circumferential and axial chest wall forces at right angles to the ribs. Nonlinear quadratic models were used to relate rib height and rib thickness to rib level, and intercostal muscle force to vector stress. Intercostal muscle force was also related to vector stress using Pearson correlation. For comparison, rib height and thickness were measured on CT scans of children. Rib height increased with rib level, increasing by 13% between the 3rd and 7th rib levels, where the 7th/8th rib was the widest part or “equator” of the rib cage, P < 0.001 (t‐test). Rib thickness showed a statistically significant 23% increase between the 3rd and 7th ribs, P = 0.004 (t‐test). Intercostal muscle force was significantly related to vector stress, Pearson correlation r = 0.944, P = 0.005. The three nonlinear quadratic models developed all had statistically significant parameter estimates with P < 0.03. External rib morphology, in particular rib height and thickness, can be predicted using statistical mathematical models. Rib height is significantly related to the calculated intercostal muscle force, showing that environmental factors affect external rib morphology. Clin. Anat. 28:512–519, 2015. © 2015 Wiley Periodicals, Inc.