Patellofemoral Pain Group (PPG)--a review of the first 100 patients to complete the course at the Regional Rehabilitation Unit Gütersloh.

Anterior knee pain syndrome is extremely common in the military and has previously often led to medical discharge. The Patellofemoral Pain Group programme of exercises developed at the Defence Medical Rehabilitation Centre at Headley Court has become the first line treatment strategy for this problem in the military. The introduction of this low level, impact free, progressive exercise programme at the Regional Rehabilitation Unit in Gutersloh has also proved useful, leading to improvements in employability of affected soldiers and reduction in symptoms.

Construction, production, and characterization of humanized anti-Lewis Y monoclonal antibody 3S193 for targeted immunotherapy of solid tumors.

The Lewis Y (Ley) antigen is a blood group-related antigen that is expressed in a high proportion of epithelial cancers (including breast, colon, ovary, and lung cancer) and is an attractive target for monoclonal antibody-directed therapy. The murine monoclonal 3S193 (IgG3) was generated in BALB/c mice by immunization with Ley-expressing cells of the MCF-7 breast carcinoma cell-line. The murine 3S193 showed high specificity for Ley in ELISA tests with synthetic Ley and Ley-containing glycoproteins and glycolipids and also reacted strongly in rosetting assays and cytotoxic tests with Ley-expressing cells. We generated a humanized form of the murine 3S193 antibody by linking cDNA sequences encoding the variable region of murine 3S913 with frameworks of the human KOL heavy chain and REI K chain. The genes for the humanized 3S193 monoclonal antibody IgG1 were transfected into mouse myeloma NS0 cells and cloned for the establishment of high antibody-producing colonies. Humanized 3S193 antibody was subsequently produced through in vitro culture and under good manufacturing practice conditions using hollow-fiber bioreactors. The purified humanized 3S193 (hu3S193) was subsequently characterized and validated for use in preliminary immunotherapy investigations. hu3S193 reacted specifically with Ley antigen, with similar avidity to the murine form. hu3S193 demonstrated potent immune effector function, with higher antibody-dependent cell-mediated cytotoxicity than its murine counterpart and potent complement-dependent cytotoxicity (ED50, 1.0 microg/ml). The in vivo immunotherapeutic potential of hu3S193 was assessed in a human breast xenograft model using MCF-7, Ley-positive cells. Six i.v. doses of up to 1 mg of hu3S193 were administered to animals bearing established tumors (120-130 mm3) with no significant effect on tumor growth. In contrast, in an MCF-7 xenograft preventive model, a 1-mg hu3S193 dosage schedule was able to significantly slow tumor growth compared with placebo and isotype-matched control IgG1 antibody. hu3S193 has promise for immunotherapy of Ley-positive tumors and is currently entering Phase I clinical trials.

Expression and characterisation of single-chain antibody fragments produced in transgenic plants against the organic herbicides atrazine and paraquat.

Single-chain antibody fragments (scAbs), which have a human C-kappa constant domain and a hexa-histidine tail attached to the carboxy terminus of the single-chain Fv (ScFv) fragments to facilitate purification, have been raised against the herbicides paraquat and atrazine and expressed in transgenic Nicotiana tabacum cv. Samsun NN. Prior to purification, the anti-atrazine scAb is expressed as up to 0.014% of soluble leaf protein and has a binding profile in ELISA, against an atrazine-bovine serum albumin (BSA) conjugate, similar to that of the scAb produced in Escherichia coli. Competition ELISA has shown that the plant-derived scAb also recognises free atrazine. Following antibody affinity purification to isolate dimers, the affinity for immobilised antigen approaches that of the parental monoclonal antibody. This was confirmed by surface plasmon resonance analysis. The purified scAb also recognises related triazine herbicides. When isolated from cell-suspension cultures, the anti-paraquat scAb binds to a paraquat conjugate in a concentration-dependent manner, with a profile similar to the parental monoclonal antibody. This is the first demonstration that functional scAbs against organic pollutants can be produced in transgenic plants and that the scAbs may be appropriate for the development of immunoassay-based detection systems.

Humanized antibodies.

Hybridoma technology enabled rodent monoclonal antibodies to be created against human pathogens and cells, but these had limited clinical utility. Protein engineering, reviewed her by Greg Winter and William Harris, is now generating antibodies for treatment of infectious disease, autoimmune disease and cancer by 'humanizing' rodent antibodies. Humanized antibodies have improved pharmacokinetics, reduced immunogenicity and have been used to clinical advantage.

Spontaneous assembly of bivalent single chain antibody fragments in Escherichia coli.

The ability of immunoglobulin Fab and single chain (ScFv) fragments to penetrate effectively into tissue from the vascular system has made these molecules excellent candidates as drug delivery systems and imaging tools. This study investigates the use of single chain antibody fragment bacterial expression vectors as a possible strategy for the production of these molecules. We have modified the pSW1-VHD1.3-VKD1.3-TAG1 vector [Ward et al. (1989) Nature 341, 544-546] which originally, when expressed in E. coli, produced an Fab fragment. In an effort to improve the affinity of the parent vector product a novel single chain antibody construct which encodes a protein with anti-P. aeruginosa activity was generated using a 14 amino acid linker [Chaudhary et al. (1990) Proc. natn. Acad. Sci. U.S.A. 87, 1066-1070]. In addition to the heavy and light chain variable domain genes, our construct also contained the light chain kappa constant domain gene to aid purification of the fragments. To underline this difference from the conventional ScFv fragment we have described this protein as a ScAb. The ScAb generated had an antigen binding capacity similar to the parent anti-P. aeruginosa antibody but was superior to the recombinant anti-P. aeruginosa Fab fragment. On HPLC and non-denaturing gel electrophoresis analysis, the ScAb was found to exist in multimeric forms while the Fab fragment existed only as a single unit. Dimeric ScAb had a similar antigen binding profile to the parent antibody.

Production of soluble single-chain T-cell receptor fragments in Escherichia coli trxB mutants.

Antibodies and T cell receptors (TCR) both belong to the immunoglobulin superfamily whose members are characterised by the possession of one or more immunoglobulin domains. The production of soluble single chain antibody fragments in Escherichia coli has, in recent years, become a routine laboratory procedure. In contrast, the production of T cell receptors in bacteria has remained problematic as the majority of the recombinant protein is insoluble. In this paper we show that single chain TCR produced in E. coli BL21 (DE3) and directed to the periplasm was also insoluble and that this was in part due to the failure of the cell protein processing machinery to cleave the pelB leader sequence. This problem was overcome by expressing the single chain TCR in the cytoplasm of E. coli which carry an inactive thioredoxin reductase gene. This strain allows the formation of disulphide bonds in the cell cytoplasm which we believe encourages the correct folding of the recombinant protein. We have constructed both a human and mouse single chain TCR in these bacteria and demonstrated using BIAcore technology that these molecules have folded in a conformation which allows their recognition by conformational specific ligands. In addition, we have used one of our soluble single chain TCR preparations to isolate a TCR specific Fab molecule from a phage antibody library.

Sequences of heavy and light chain variable regions from four bovine immunoglobulins.

Oligodeoxyribonucleotide primers based on the 5' ends of bovine IgG1/2 and lambda constant (C) region genes, together with primers encoding conserved amino acids at the N-terminus of mature variable (V) regions from other species, have been used in cDNA and polymerase chain reactions (PCRs) to amplify heavy and light chain V region cDNA from bovine heterohybridomas. The amino acid sequences of VH and V lambda from four bovine immunoglobulins of different specificities are presented.

Cloning and expression in Escherichia coli of the cDNA encoding human cardiac troponin I.

We have cloned and sequenced the human cardiac troponin I (cTnI)-encoding cDNA with the aim of expressing the cDNA in Escherichia coli. The cDNA was successfully expressed as a fusion product with beta-galactosidase and as an unfused protein. Both polypeptides were recognised by an anti-human cTnI antibody.

Production of a functional single chain antibody attached to the surface of a plant virus.

A potato virus X (PVX) vector was used to express a single chain antibody fragment (scFv) against the herbicide diuron, as a fusion to the viral coat protein. The modified virus accumulated in inoculated Nicotiana clevelandii plants and assembled to give virus particles carrying the antibody fragment. Electron microscopy was used to show that virus particles from infected leaf sap were specifically trapped on grids coated with a diuron-BSA conjugate. The results demonstrate that the PVX vector can be used as a presentation system for functional scFv.

Newly synthesised DNA in ageing human cells in culture treated with 4-nitroquinoline-1-oxide.

Two lines of normal human embryonic lung fibroblasts, MRC-5 and F2002, were serially subcultured until senescence was attained. When cells were exposed to varying concentrations of the chemical carcinogen 4-nitroquinoline-1-oxide (4-NQO), the rate of DNA synthesis (as measured by thymidine incorporation) was reduced in a dose-dependent fashion in cells from both early and late passages. While the overall amount of incorporation was considerably lower in old cells, the extent of inhibition caused by 4-NQO treatment (relative to appropriate controls) was not related to culture age. Alkaline sucrose density gradient analysis of newly synthesised DNA from cells pre-treated with 4-NQO failed to detect any significant variation in the size of labelled DNA from cells examined immediately after incubation with radioactive thymidine. The shift of this labelled material to high molecular weight in 4-NQO-treated cells also showed no age-related difference.

Colorimetric method for detecting amplified nucleic acids.

Methods for detecting and measuring the success of nucleic acid sequence amplifications can be developed by detecting the by-products of amplification procedures. One method includes the detection of inorganic phosphate (Pi) during or on completion of the PCR. The method requires modification of assay conditions to prevent thermal- and template-independent enzymatic activity from nonspecifically hydrolyzing dNTPs. Detection of Pi by the traditional Fiske-SubbaRow method provides a sensitivity similar to ethidium bromide staining of amplified products. The method offers a simple and rapid assay for amplified nucleic acids and can be useful in assays where confirmation of the amplified DNA product is not essential.

Simultaneous detection of microorganisms in soil suspension based on PCR amplification of bacterial 16S rRNA fragments.

The effect of buffer composition on simultaneous PCR amplification of 16S rRNA gene fragments of five bacterial species was examined using a number of different buffer systems. Tris-based PCR buffers at final concentrations of 10 mM proved unreliable. However, when the final concentration of Tris was increased to 75 mM, all five samples were routinely detected. The use of other buffers, 3-[(1,1-dimethyl-2-hydroxyethyl)amino]-2-hydroxypropanesulfonic acid (AMPSO) and 3-[cyclohexylamino]-2-hydroxy-1-propanesulfonic acid (CAPSO), resulted in PCR amplification of five products even at low final concentrations (10 mM). The presence of certain proteins in the amplification reaction could overcome an inhibitory effect seen when soil suspension was present in the reaction, as might occur when testing field samples for the presence of bacteria. Bovine serum albumin was found to be the most effective additive tested in overcoming inhibition.

Uterine dehiscence following laparoscopic myomectomy.

BACKGROUND: Laparoscopic myomectomy is a new procedure that is growing in popularity. The natural history of pregnancy following laparoscopic myomectomy is unknown. CASE: A 24-year-old white woman, gravida 0, with infertility and endometriosis, conceived after a laparoscopic procedure that included myomectomy. At 34 weeks' gestation, the patient experienced uterine dehiscence at the site of myomectomy. An emergency cesarean delivery was performed and the uterus was oversewn. Both mother and infant had satisfactory hospital courses. CONCLUSION: Meticulous closure of the myometrial bed following myomectomy is difficult via the laparoscope, and this could interfere with the integrity of the scar. If further studies confirm this experience, then laparoscopic myomectomy may need to be limited to patients who do not desire further childbearing.

Initial experience with the Boyle uterine elevator.

Since 1990, our institution has used the Boyle uterine elevator in 57 abdominal hysterectomies. The device aids in retraction of the uterus and minimizes the use of self-retaining retractors and bowel packing. The uterine elevator also aids in dissection of the bladder and incision of the cardinal and uterosacral ligaments. The mean hospital stay has been reduced to 2.6 days. Postoperative complications are rare. We conclude that this device is a useful adjunct in the performance of abdominal hysterectomy.

The isolation of super-sensitive anti-hapten antibodies from combinatorial antibody libraries derived from sheep.

The complexity and expense of producing anti-hapten monoclonals via the traditional hybridoma route and the preferential selection of antibodies that recognise the conjugated form of the hapten, over antibodies that specifically recognise free hapten, are two of the more important problems that have limited the development and application of anti-hapten antibodies. The advent of phage display technology allows the rapid isolation of monoclonal antibody fragments from libraries of different antibodies (>10(8)) displayed on the surface of filamentous bacteriophages. Much of the power of this new approach lies in the flexibility with which these libraries can be screened for suitable binders. Using an optimised selection procedure, we have isolated from a sheep antibody phage display library, super-sensitive anti-hapten antibodies specific for the herbicide and environmental pollutant, atrazine. In particular, two phage clones have been isolated that can be expressed cheaply and in quantity in Escherichia coli, demonstrate excellent stability in nonphysiological conditions and are exciting prospects for immunoassay applications including ELISA, dip-stick formats, on-line monitoring and biosensor technologies. In ELISA formats they show low levels of cross reactivity with related molecules and a limit of detection of a 1-2 parts per trillion (p.p.t.), well within the 100 p.p.t. required by EC legislation.

Binding characteristics of anti-atrazine monoclonal antibodies and their fragments synthesised in bacteria and plants.

Single-chain antibody fragments (scAb), specific for the herbicide atrazine, have been expressed in the bacterium Escherichia coli and in transgenic tobacco plants. The scAb could be purified as a monomer (monovalent) via a hexa-histidine tail or as a dimer (divalent) by antibody affinity chromatography. In competition ELISA, the bacterial scAb showed the same specificity for atrazine and related triazine herbicides as the parental mAb cell line, but both plant and bacterial monomeric scAbs showed increased sensitivity to free atrazine. Surface plasmon resonance (BIAcore 2000) analysis confirmed that purified scAb, derived from plant or bacteria, retained similar association rates as the mAb. However, the monomeric plant and bacterial scAbs showed a lower affinity for immobilised antigen, than the equivalent dimeric scAbs or mAb. This decrease in affinity was due to a 10 fold slower dissociation rate and is likely due to loss of the avidity contribution of dimeric molecules.

Expression of foreign genes in mammalian cells using an antibody fusion system.

An expression system is described whereby a gene product is expressed fused to an antibody Fab fragment to form an antibody-like molecule. The antigen binding function of the original antibody is retained and the foreign gene replaces the CH2 and CH3 regions of the heavy chain. The fusion protein is secreted as if it were an antibody, and can be purified using the antigen-binding function of the Fab-like part of the molecule. In principle, any open reading frame can be expressed and it is not necessary to develop an individual purification scheme, or any analytical reagents such as antibodies, for the expressed protein, as both these functions can be performed by the Fab part of the fusion protein. In practice, the nature of the nonantibody part of the fusion influences the efficiency of expression and secretion, and detailed guidance is given on trouble-shooting and maximizing expression.

Retention of neutralising activity by recombinant anti-pneumolysin antibody fragments.

The variable domains of a neutralising (prevents erythrocyte lysis) anti-pneumolysin monoclonal antibody have been cloned and expressed as functional protein in Escherichia coli. Purification of the anti-pneumolysin single-chain antibody fragment, via antibody-affinity or metal-chelate affinity chromatography, resulted in product that was predominantly in a dimeric or monomeric form, respectively. The dimeric single-chain antibody fragment showed a higher sensitivity and affinity for immobilised antigen in both ELISA and BIAcore studies. The dimeric single-chain antibody fragment was as effective at protecting erythrocytes from lysis as the parent monoclonal. The monomeric, low affinity single-chain antibody fragment, showed reduced neutralising potency. As antibiotic resistant Streptococcus pneumoniae strains continue to show an increasing word-wide distribution, recombinant, neutralising antibody fragments, may provide an additional class of molecules useful in the treatment of toxaemia.

Use of corticosteroids as an adjuvant in terminal salpingostomy.

Between the years 1977 and 1980, 26 women underwent terminal salpingostomy with a standard operative procedure at Vanderbilt University Medical Center. The term pregnancy rate was 23%, and the ectopic pregnancy rate was 13%. Fourteen patients received dexamethasone perioperatively; and of these, 3 had term pregnancies (21%). Of the 12 patients not receiving dexamethasone, there were also three term pregnancies (25%). It is concluded from this small series that perioperative corticosteroids do not improve the term pregnancy rate for terminal salpingostomy.

Expression of foreign genes in Mammalian cells using an antibody fusion system.

Whereas the expression of foreign genes in mammalian cells usually proves successful, the purification of gene products is often a difficult and time-consuming process. The availability of monoclonal antibodies to the foreign protein can greatly assist in small scale purification, but where antibodies are not available, alternatives have to be sought One useful approach involves the fusion of the foreign gene adjacent to a gene segment encoding an antibody heavy chain variable region (1). By transfection of this construct into a cell line producing a compatible light chain or by cotransfection of the fusion product with a light chain gene, an antibody-like molecule can be produced and purified using the corresponding antigen.