Background

Phage-display panning is an integral part of biomedical research. Regular
panning methods are sometimes complicated by inefficient detachment of the
captured phages from the antigen-coated solid supports, which prompted us to
modify. Here, we produce an efficient antigen-specific single chain fragment
variable (scFv) antibody by using a target-related molecule that favored
selection ofrecombinant antibodies.

To produce more selective and specific anti-idiotypic scFv-antibodies from a
cDNA library, constructed from HM-1 killer toxin (HM-1)-neutralizing monoclonal
antibodies (nmAb-KT), the method was modified by using an elution buffer
supplemented with HM-1 that shares structural and functional similarities with
the active site of the scFv antibody. Competitive binding of HM-1 to nmAb-KT
allowed easy and quick dissociation of scFv-displayed phages from immobilized
nmAb-KT to select specific anti-idiotypic scFv antibodies of HM-1. After
modified panning, 80% clones (40/50) showed several times higher binding
affinity to nmAb-KT than regular panning. The major populations (48%) of these
clones (scFv K1) were genotypically same and had strong cytocidal activity
against Saccharomyces and Candida species. The scFv K1
(K_d value = 4.62 × 10^-8 M) had strong reactivity
toward nmAb-KT, like HM-1 (K_d value = 6.74 × 10^-9
M) as judged by SPR analysis.

The scFv antibodies generated after modified subtractive panning appear to
have superior binding properties and cytocidal activity than regular panning. A
simple modification of the elution condition in the phage-display panning
protocol makes a large difference in determining success. Our method offers an
attractive platform to discover potential therapeutic candidates.

Background
We aimed to study the effects of intra-articular injection of jellyfish mucin (qniumucin) on articular cartilage degeneration in a model of osteoarthritis (OA) created in rabbit knees by resection of the anterior cruciate ligament. Qniumucin was extracted from Aurelia aurita (moon jellyfish) and Stomolophus nomurai (Nomura's jellyfish) and purified by ion exchange chromatography. The OA model used 36 knees in 18 Japanese white rabbits. Purified qniumucin extracts from S. nomurai or A. aurita were used at 1 mg/ml. Rabbits were divided into four groups: a control (C) group injected with saline; a hyaluronic acid (HA)-only group (H group); two qniumucin-only groups (M groups); and two qniumucin + HA groups (MH groups). One milligram of each solution was injected intra-articularly once a week for 5 consecutive weeks, starting from 4 weeks after surgery. Ten weeks after surgery, the articular cartilage was evaluated macroscopically and histologically.

Results
In the C and M groups, macroscopic cartilage defects extended to the subchondral bone medially and laterally. When the H and both MH groups were compared, only minor cartilage degeneration was observed in groups treated with qniumucin in contrast to the group without qniumucin. Histologically, densely safranin-O-stained cartilage layers were observed in the H and two MH groups, but cartilage was strongly maintained in both MH groups.

Conclusion
At the concentrations of qniumucin used in this study, injection together with HA inhibited articular cartilage degeneration in this model of OA.

Background
Recombinant protein expression in mammalian cells is mostly achieved by stable integration of transgenes into the chromosomal DNA of established cell lines. The chromosomal surroundings have strong influences on the expression of transgenes. The exploitation of defined loci by targeting expression constructs with different regulatory elements is an approach to design high level expression systems. Further, this allows to evaluate the impact of chromosomal surroundings on distinct vector constructs.

Results
We explored antibody expression upon targeting diverse expression constructs into previously tagged loci in CHO-K1 and HEK293 cells that exhibit high reporter gene expression. These loci were selected by random transfer of reporter cassettes and subsequent screening. Both, retroviral infection and plasmid transfection with eGFP or antibody expression cassettes were employed for tagging. The tagged cell clones were screened for expression and single copy integration. Cell clones producing > 20 pg/cell in 24 hours could be identified. Selected integration sites that had been flanked with heterologous recombinase target sites (FRTs) were targeted by Flp recombinase mediated cassette exchange (RMCE). The results give proof of principle for consistent protein expression upon RMCE. Upon targeting antibody expression cassettes 90-100% of all resulting cell clones showed correct integration. Antibody production was found to be highly consistent within the individual cell clones as expected from their isogenic nature. However, the nature and orientation of expression control elements revealed to be critical. The impact of different promoters was examined with the tag-and-targeting approach. For each of the chosen promoters high expression sites were identified. However, each site supported the chosen promoters to a different extent, indicating that the strength of a particular promoter is dominantly defined by its chromosomal context.

Conclusion
RMCE provides a powerful method to specifically design vectors for optimized gene expression with high accuracy. Upon considering the specific requirements of chromosomal sites this method provides a unique tool to exploit such sites for predictable expression of biotechnologically relevant proteins such as antibodies.

Background
MicroRNAs are small non-coding RNAs that post-transcriptionally regulate gene expression and their expression is frequently altered in human diseases, including cancer. To correlate clinically relevant parameters with microRNA expression, total RNA is frequently prepared from samples that were archived for various time periods in frozen tissue banks but, unfortunately, RNA integrity is not always preserved in these frozen tissues. Here, we investigate whether experimentally induced RNA degradation affects microRNA expression profiles.

Results
Tissue samples were maintained on ice for defined time periods prior to total RNA extraction, which resulted in different degrees of RNA degradation. MicroRNA expression was then analyzed by microarray analysis (miCHIP) or microRNA-specific real-time quantitative PCR (miQPCR). Our results demonstrate that the loss of RNA integrity leads to in unpredictability of microRNA expression profiles for both, array-based and miQPCR assays.

Conclusion
MicroRNA expression cannot be reliably profiled in degraded total RNA. For the profiling of microRNAs we recommend use of RNA samples with a RNA integrity number equal to or above seven.

Background

Human umbilical cord blood-derived unrestricted somatic stem cells (USSCs),
which are capable of multilineage differentiation, are currently under
investigation for a number of therapeutic applications. A major obstacle to
their clinical use is the fact that in vitro expansion is still
dependent upon fetal calf serum, which could be a source of pathogens. In this
study, we investigate the capacity of three different stem cell culture media to
support USSCs in serum-free conditions; HEScGRO™, PSM and USSC growth
medium^ACF. Our findings demonstrate that USSCs do not grow in
HEScGRO™ or PSM, but we were able to isolate, proliferate and maintain
multipotency of three USSC lines in USSC growth medium^ACF.

For the first one to three passages, cells grown in USSC growth
medium^ACF proliferate and maintain their morphology, but with
continued passaging the cells form spherical cell aggregates. Upon dissociation
of spheres, cells continue to grow in suspension and form new spheres.
Dissociated cells can also revert to monolayer growth when cultured on
extracellular matrix support (fibronectin or gelatin), or in medium containing
fetal calf serum. Analysis of markers associated with pluripotency (Oct4 and
Sox2) and differentiation (FoxA2, Brachyury, Goosecoid, Nestin, Pax6,
Gata6 and Cytokeratin 8) confirms that cells in the spheres maintain their
gene expression profile. The cells in the spheres also retain the ability to
differentiate in vitro to form cells representative of the three
germline layers after five passages.

These data suggest that USSC growth medium^ACF maintains USSCs in
an undifferentiated state and supports growth in suspension. This is the first
demonstration that USSCs can grow in a serum- and animal component-free medium
and that USSCs can form spheres.