Development of a novel mammalian display system for selection of antibodies against membrane proteins.

Reliable, specific polyclonal and monoclonal antibodies are important tools in research and medicine. However, the discovery of antibodies against their targets in their native forms is difficult. Here, we present a novel method for discovery of antibodies against membrane proteins in their native configuration in mammalian cells. The method involves the co-expression of an antibody library in a population of mammalian cells that express the target polypeptide within a natural membrane environment on the cell surface. Cells that secrete a single-chain fragment variable (scFv) that binds to the target membrane protein thereby become self-labeled, enabling enrichment and isolation by magnetic sorting and FRET-based flow sorting. Library sizes of up to 109 variants can be screened, thus allowing campaigns of naïve scFv libraries to be selected against membrane protein antigens in a Chinese hamster ovary cell system. We validate this method by screening a synthetic naïve human scFv library against Chinese hamster ovary cells expressing the oncogenic target epithelial cell adhesion molecule and identify a panel of three novel binders to this membrane protein, one with a dissociation constant (KD ) as low as 0.8 nm We further demonstrate that the identified antibodies have utility for killing epithelial cell adhesion molecule-positive cells when used as a targeting domain on chimeric antigen receptor T cells. Thus, we provide a new tool for identifying novel antibodies that act against membrane proteins, which could catalyze the discovery of new candidates for antibody-based therapies.

The impact of thioredoxin reduction of allosteric disulfide bonds on the therapeutic potential of monoclonal antibodies.

Therapeutic mAbs are used to manage a wide range of cancers and autoimmune disorders. However, mAb-based treatments are not always successful, highlighting the need for a better understanding of the factors influencing mAb efficacy. Increased levels of oxidative stress associated with several diseases are counteracted by the activities of various oxidoreductase enzymes, such as thioredoxin (Trx), which also reduces allosteric disulfide bonds in proteins, including mAbs. Here, using an array of in vitro assays, we explored the functional effects of Trx-mediated reduction on the mechanisms of action of six therapeutic mAbs. We found that Trx reduces the interchain disulfide bonds of the mAbs, after which they remain intact but have altered function. In general, this reduction increased antigen-binding capacity, resulting in, for example, enhanced tumor necrosis factor (TNF) neutralization by two anti-TNF mAbs. Conversely, Trx reduction decreased the antiproliferative activity of an anti-tyrosine kinase-type cell-surface receptor HER2 mAb. In all of the mAbs, Fc receptor binding was abrogated by Trx activity, with significant loss in both complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (ADCC) activity of the mAbs tested. We also confirmed that without alkylation, Trx-reduced interchain disulfide bonds reoxidize, and ADCC activity is restored. In summary, Trx-mediated reduction has a substantial impact on the functional effects of an mAb, including variable effects on antigen binding and Fc function, with the potential to significantly impact mAb efficacy in vivo.

Surrogate CD16-expressing effector cell lines for determining the bioactivity of therapeutic monoclonal antibodies.

Traditional antibody dependent cellular cytotoxicity (ADCC) assays use donor derived natural killer (NK) or peripheral blood mononuclear cells, but donor genetic variability and the technically challenging nature of the assay means that alternative in vitro assay formats are required. We explored the utility of two reporter gene cell lines, the J2 and J9, as surrogate effector cells for ADCC assays. Both express the ADCC relevant Fcγ receptor CD16, crosslinking of which leads to firefly luciferase expression. For anti-CD20 rituximab and anti-HER2 trastuzumab (both IgG1 monoclonal antibodies, mAbs) a dose dependent firefly luciferase response was observed exclusively in the presence of their respective targets, representing the molecular interaction which potentiates ADCC activity. Importantly, both surrogate effector and NK cell based assays gave statistically similar values for rituximab ADCC activity. Increased engagement with target cell bound mAbs was determined to be cytotoxic for the J2 and J9 cell lines at the assay end point (at which luciferase expression is measured). However, use of the J9 cells containing the constitutively expressed renilla luciferase gene enabled data normalisation and corrected for fluctuations in both cell number and viability providing an advantage over currently available surrogate effector cell-lines. Abrogated ADCC activity with IgG4 mAbs, but enhanced activity with an IgG1 non-fucosylated mAb, was seen with the J9 cell line, as expected. Additionally, two rituximab products (biosimilars in development) with similar binding by flow cytometry, N-glycan profiles using HPLC and CD16 binding by surface plasmon resonance showed comparable ADCC activity to Mabthera. The ADCC activity of another anti-CD20 mAb, ofatumumab, reported only with primary cell based assays to date was also measured. This is the first report of a dual reporter gene based ADCC assay.

Protective effect of human recombinant copper-zinc superoxide dismutase (hr-cuznsod) on intermediate burn survival in rats.

Objectives. Superoxide dismutase, acting as a scavenger of oxygen free radicals, has shown mixed results in increasing survival from burn wounds. We previously demonstrated that human recombinant copper-zinc superoxide dismutase could increase the survival of failing ischaemic flaps in a rat model. Because of the similar pathophysiology of tissue ischaemia in flaps and intermediate zone burns, we conducted a later study employing two groups of rats with standardized intermediate burns, to ascertain whether or not human recombinant copper-zinc superoxide dismutase could increase intermediate burn zone survival in rats. The results showed that post-burn human recombinant copper-zinc superoxide dismutase failed to improve intermediate burn zone survival. We decided to undertake a new study to ascertain whether there was a protective effect of human recombinant copper-zinc superoxide dismutase in intermediate burns. Methods. This controlled study employed two groups of rats, one of which received prophylactic treatment with human recombinant copper-zinc superoxide dismutase before the induction of standardized intermediate burns. Results. The results showed that pre-burn human recombinant copper-zinc superoxide dismutase also failed to improve intermediate burn zone survival. Conclusions. Further studies are needed to fully understand the effect of oxygen free radicals in burn wound pathophysiology and to determine whether human recombinant copper-zinc superoxide dismutase has a place in the clinical management of burns.

Burns and diabetes.

Diabetes is often considered a risk factor for poor wound healing and increased complication rates for plastic surgery procedures. Burn injury in diabetic patients may have implications for the length of stay and number of operations required. We therefore we examined the characteristics of diabetic patients admitted to our burn unit and the impact of their condition on their hospital course. Charts of all patients with diabetes admitted to the burn unit from 1995 to 2000 were reviewed (n = 73). Demographic data, percent body surface area burned, anatomical location of the burn, number of surgical procedures required, length and cost of stay, and outcome were noted. The control population included 150 consecutive patients without diabetes treated during the same period. Diabetic patients were older and underwent a higher number of surgical procedures, with increased length of stay and increased mortality, despite an equivalent body surface area burned. They had a higher incidence of scald burns in the lower extremities than the non-diabetic population. This work shows that diabetic patients constitute a unique group. They are significantly older, have an increased rate of surgical interventions, increased hospital stay, and significantly increased mortality compared to a control group with similar surface area burns. This group is also more likely to have scald burns in the lower extremities, mostly due to diabetic neuropathy. This work emphasizes the importance of education and prevention programmes directed towards this group of patients, in order to decrease morbidity, mortality, and hospital costs.

Excision of group II introns as circles.

Group II introns are usually removed from precursor RNAs as lariats comprised of a circular component and a short 3' tail. We find that group II introns can also be excised as complete circles. Circle formation requires release of the 3' exon of a splicing substrate, apparently by a trans splicing mechanism. After 3' exon release, the terminal uridine of the intron attacks the 5' splice site, releasing the 5' exon and joining the first and last intron residues by a 2'-5' phosphodiester bond. RNA isolated from yeast mitochondria also contains circles, indicating that at least one group II intron (aI2) forms circles in vivo. Furthermore, analysis of RNA and DNA from certain mutant yeast strains shows that circular DNA introns exist and are produced by reverse transcription of RNA, rather than by ectopic homing.

Effect of high dose and low dose aspirin on survival of random pattern flaps in rats.

This paper studies which of the physiological effects of aspirin is responsible for increasing the survival of random flaps in rats, found in an earlier experiment. We wished to confirm that the antiaggregating--antithrombotic effect was responsible for the increased survival of flaps without microvascular anastomosis. Three groups of rats with standardised random pattern flaps were used. The first two were given aspirin 200 mg/kg (high dose, n = 27) or 40 mg/kg (low dose, n = 21) and the third (n = 28) acted as controls. The beneficial effects of aspirin were restricted to the high dose group. Since the low dose group also showed antiaggregation of platelets, but without the anti-inflammatory or vasodilatory effects, the results indicate that the antiaggregating effect alone was not responsible for the increased survival of the flaps.

Intraoperative positioning for circumferential extremity burns.

A technique for intra-operative patient positioning is described which allows circumferential access to burned extremities for prepping and surgery. This technique avoids the need for turning the patient during the operative procedure. In our 13 yr experience, there have been no complications attributed to this technique.

Acellular allograft dermal matrix: immediate or delayed epidermal coverage?

In a prospective, randomized study seventeen patients received skin grafts to a freshly excised burn wound. One group was grafted with a deantigenized dermal matrix and immediately overgrafted with thin autograft. The second group was grafted with dermal matrix, which was then covered with bank allograft for protection, and autografted 1 week later. Each group also received a standard split thickness control graft. Assessment was carried out for up to 1 year. There were no statistically significant differences of graft take between any of the groups, or in the Vancouver scar score at follow-up. Thin donor sites used for dermal matrix coverage healed faster than standard control graft sites, P<0.001. Immediate grafting of acellular dermal matrix with thin autograft works well and leads to an acceptable late result, with faster donor site healing than standard split thickness grafts.

Analysis of the M-shell spectra emitted by a short-pulse laser-created tantalum plasma

The spectrum of tantalum emitted by a subpicosecond laser-created plasma, was recorded in the regions of the 3d-5f, 3d-4f, and 3d-4p transitions. The main difference with a nanosecond laser-created plasma spectrum is a broad understructure appearing under the 3d-5f transitions. An interpretation of this feature as a density effect is proposed. The supertransition array model is used for interpreting the spectrum, assuming local thermodynamic equilibrium (LTE) at some effective temperature. An interpretation of the 3d-4f spectrum using the more detailed unresolved transition array formalism, which does not assume LTE, is also proposed. Fitted contributions of the different ionic species differ slightly from the LTE-predicted values.

Tri-partite assay for studying exon ligation by the ai5gamma group II intron.

Group II introns self-splice via a two-step mechanism: cleavage at the 5' splice site followed by exon ligation at the 3' splice site. The second step has been difficult to study in vitro because it is generally faster than the first. Herein we describe development and partial kinetic characterization of a novel assay for studying the second step in isolation. In this system, a truncated linear intron (nucleotides 1-881) mediates exon ligation between two oligonucleotide substrates: a 19 nt 5' exon and a 3' substrate consisting of the last 6 nucleotides of the intron plus a 6 nucleotide 3' exon. We found that neither the exact structure of domain 6 nor the identity of nucleotides flanking the 3' splice site is critical for accurate 3' splice site choice by the ai5gamma group II intron. The multiple turnover k(cat) (0.14 min(-)(1)) is slower than the single turnover k(obs) (0.6-0.7 min(-)(1)), consistent with rate-limiting product release under steady-state conditions. Decreased single turnover rates at lower pHs were more consistent with loss of catalytic activity than with rate-limiting chemistry. Binding of the 3' substrate (K(m) = 2.6 microM) could be improved by changing a long-range A:U base pair involving the last intronic nucleotide (the gamma-gamma' interaction) to G:C (K(m(3)(')(substrate)) = 1 microM).

Inflammatory fibroid polyp and Helicobacter pylori. Aetiology or coincidence?

OBJECTIVE: To evaluate our incidence of inflammatory fibroid polyps, compare our experience with that of others, and to analyze the possible pathophysiological and aetiological factors. DESIGN: Retrospective review. SETTING: Teaching hospital. MATERIAL: All histopathological slides of the gastrointestinal tract. MAIN OUTCOME MEASURES: Incidence and treatment in our Medical Center and elsewhere. RESULTS: We could find only one case of inflammatory fibroid polyp, an estimated incidence of 1/4000. Between 1987-1996 only 331 were reported elsewhere, most of which (293, 88.5%), were located in the stomach. CONCLUSION: Primary mucosal damage can expose the stroma to several irritants (chemical, mechanical and biological), that may subsequently cause inflammatory fibroid polyps in certain people.

Quantitative trait locus mapping in dairy cattle by means of selective milk DNA pooling using dinucleotide microsatellite markers: analysis of milk protein percentage.

"Selective DNA pooling" accomplishes quantitative trait locus (QTL) mapping through densitometric estimates of marker allele frequencies in pooled DNA samples of phenotypically extreme individuals. With poly(TG) microsatellites, such estimates are confounded by "shadow" ("stutter") bands. A correction procedure was developed on the basis of an observed linear regression between shadow band intensity and allele TG repeat number. Using this procedure, a selective DNA pooling study with respect to milk protein percentage was implemented in Israel-Holstein dairy cattle. Pools were prepared from milk samples of high and low daughters of each of seven sires and genotyped with respect to 11 markers. Highly significant associations with milk protein percentage were found for 5 of the markers; 4 of these markers confirmed previous reports. Selective DNA pooling accessed 80.6 and 48.3%, respectively, of the information that would have been available through individual selective genotyping or total population genotyping. In effect, the statistical power of 45,600 individual genotypings was obtained from 328 pool genotypings. This methodology can make genome-wide mapping of QTL accessible to moderately sized breeding organizations.

Prevention of needle-stick injury by the scooping-resheathing method.

BACKGROUND: The objective of this study was to determine the effects of teaching the scooping-resheathing method on the incidence of needle-stick injuries in medical students. METHODS: Before starting their first clerkship, 81 medical students were given a 15-min lecture on the high incidence and dangers of needle-stick injuries and a demonstration of the scooping-resheathing method. The number of needle-stick injuries that occurred during the 3-month clerkship was compared with the number reported by 86 medical students who had completed their first clerkship 1 year previously and had not been given such instruction. RESULTS: Compared with controls, the study group had a 3.8-fold lower risk of needle-stick injury (95% confidence interval, 2.0-7.4, P < 0.0001) and a 8.3-fold lower risk of multiple needle-stick injuries (95% confidence interval, 2.0-35.0, P < 0.001). Those in the study group, who consistently used the scooping method had a much lower risk of injury than those who did not (1 of 36 [2.8%] vs. 8 of 45 [17.4%], P = 0.039). CONCLUSIONS: We conclude that a lecture recommending the scooping-resheathing method is effective in reducing the risk of needle-stick injuries in medical students during their first rotation. Because this is the first time that an intervention not requiring change in equipment has been successful, further studies are warranted to substantiate our findings and for extrapolation to other medical personnel in other cultural settings.

Primer tRNA3Lys on the viral genome exists in unextended and two-base extended forms within mature human immunodeficiency virus type 1.

In human immunodeficiency virus type 1, tRNALys3 is placed upon the primer binding site, where it serves as the primer for reverse transcription. We show herein that genomic placement occurs independently of precursor protein processing and that the placed tRNALys3 exists in two major forms: unextended or in a form extended by the first two DNA bases incorporated, C and T. The two-base extended form of the tRNALys3 is absent in a protease-negative mutant, indicating a requirement for mature viral proteins to initiate reverse transcription within the virus.

Multiple forms of tRNA(Lys3) in HIV-1.

tRNALys3 is the primer for HIV-1 reverse transcriptase (RI) and is selectively incorporated into HIV-1 during viral assembly. While whole cell extracts of uninfected or infected cells contain only one detectable form of tRNALys3, multiple forms of tRNALys3 are detected in the virus released into the cell culture media. These tRNALys3 isoacceptors are found in HIV-1 produced from newly infected cord blood lymphocytes and from cells chronically infected with HIV-1, such as the lymphocytic cell line H9 and the monocytic cell lines U937 and PLB. They can be detected through the use of either RPC-5 column chromatography of tRNA aminoacylated with radioactive lysine or northern blot analysis using a tRNALys3-specific DNA hybridization probe. Both RPC-5 chromatography and northern blot analysis show the cytoplasmic form of tRNALys3 to be the major abundance form of tRNALys3 in the virus. Starting with the viral RNA isolated from HIV (PLB), the tRNALys3 species resolved by RPC-5 into peaks 2, 3, and 4 were deacylated and 3' end-labeled by heat-annealing the RNA in each peak to synthetic HIV genomic RNA, and extending the hybridized species one base using HIV-1 RT and radioactive dCTP. An electrophoretic comparison of the partial T1 digest pattern of purified human placental tRNALys3 with those of the RPC-5 resolved species showed that the labeled RNA species in each peak was tRNALys3. These radioactive tRNALys3 species retained their relative mobilities when rechromatographed on RPC-5. When total HIV (PLB) RNA was used as the source of primer/template, and similarly extended with RT in the presence of radioactive dCTP, the major priming tRNA resolved by RPC-5 had a chromatographic mobility identical to peak 3. This tRNA primer has a T1 digest pattern identifying it as tRNALys3. These results indicate that the major tRNALys3 species present in the virus is also the major tRNALys3 isoacceptor used as the primer for reverse transcription.