Diagnostic accuracy of contemporary multidetector computed tomography (MDCT) for the detection of lumbar disc herniation.

OBJECTIVES: To evaluate the diagnostic accuracy of multidetector CT (MDCT) for detection of lumbar disc herniation with MRI as standard of reference. METHODS: Patients with low back pain underwent indicated MDCT (128-row MDCT, helical pitch), 60 patients with iterative reconstruction (IR) and 67 patients with filtered back projection (FBP). Lumbar spine MRI (1.5 T) was performed within 1 month. Signal-to-noise ratios (SNR) of cerebrospinal fluid (CSF), annulus fibrosus (AF) and the spinal cord (SC) were determined for all modalities. Two readers independently rated image quality (IQ), diagnostic confidence and accuracy in the diagnosis of lumbar disc herniation using MRI as standard of reference. Inter-reader correlation was assessed with weighted κ. RESULTS: Sensitivity, specificity, precision and accuracy of MDCT for disc protrusion were 98.8%, 96.5%, 97.1%, 97.8% (disc level), 97.7%, 92.9%, 98.6%, 96.9% (patient level). SNR of IR was significantly higher than FBP. IQ was significantly better in IR owing to visually reduced noise and improved delineation of the discs. κ (>0.90) was excellent for both algorithms. CONCLUSION: MDCT of the lumbar spine yields high diagnostic accuracy for detection of lumbar disc herniation. IR improves image quality so that the provided diagnostic accuracy is principally equivalent to MRI. KEY POINTS: • MDCT is an accurate alternative to MRI in disc herniation diagnosis. • By IR enhanced image quality improves MDCT diagnostic confidence similar to MRI. • Advances in CT technology contribute to improved diagnostic performance in lumbar spine imaging.

Automatic path proposal computation for CT-guided percutaneous liver biopsy.

PURPOSE: To evaluate feasibility of automatic software-based path proposals for CT-guided percutaneous biopsies. METHODS: Thirty-three patients (60 [Formula: see text] 12 years) referred for CT-guided biopsy of focal liver lesions were consecutively included. Pre-interventional CT and dedicated software (FraunhoferMeVis Pathfinder) were used for (semi)automatic segmentation of relevant structures. The software subsequently generated three path proposals in downward quality for CT-guided biopsy. Proposed needle paths were compared with consensus proposal of two experts (comparable, less suitable, not feasible). In case of comparable results, equivalent approach to software-based path proposal was used. Quality of segmentation process was evaluated (Likert scale, 1 [Formula: see text] best, 6 [Formula: see text] worst), and time for processing was registered. RESULTS: All biopsies were performed successfully without complications. In 91 % one of the three automatic path proposals was rated comparable to experts' proposal. None of the first proposals was rated not feasible, and 76 % were rated comparable to the experts' proposal. 7 % automatic path proposals were rated not feasible, all being second choice ([Formula: see text]) or third choice ([Formula: see text]). In 79 %, segmentation at least was good. Average total time for establishing automatic path proposal was 42 [Formula: see text] 9 s. CONCLUSION: Automatic software-based path proposal for CT-guided liver biopsies in the majority provides path proposals that are easy to establish and comparable to experts' insertion trajectories.

Carnosine treatment in combination with ACE inhibition in diabetic rats.

In humans, we reported an association of a certain allele of carnosinase gene with reduced carnosinase activity and absence of nephropathy in diabetic patients. CN1 degrades histidine dipeptides such as carnosine and anserine. Further, we and others showed that treatment with carnosine improves renal function and wound healing in diabetic mice and rats. We now investigated the effects of carnosine treatment alone and in combination with ACE inhibition, a clinically established nephroprotective drug in diabetic nephropathy. Male Sprague-Dawley rats were injected i.v. with streptozotocin (STZ) to induce diabetes. After 4 weeks, rats were unilaterally nephrectomized and randomized for 24 weeks of treatment with carnosine, lisinopril or both. Renal CN1 protein concentrations were increased under diabetic conditions which correlated with decreased anserine levels. Carnosine treatment normalized CN1 abundance and reduced glucosuria, blood concentrations of glycosylated hemoglobin (HbA1c), carboxyl-methyl lysine (CML), N-acetylglucosamine (GlcNac; all p<0.05 vs. non-treated STZ rats), reduced cataract formation (p<0.05) and urinary albumin excretion (p<0.05), preserved podocyte number (p<0.05) and normalized the increased renal tissue CN1 protein concentration. Treatment with lisinopril had no effect on HbA1C, glucosuria, cataract formation and CN1 concentration, but reduced albumin excretion rate more effectively than carnosine treatment (p<0.05). Treatment with both carnosine and lisinopril combined the effects of single treatment, albeit without additive effect on podocyte number or albuminuria. Increased CN1 amount resulted in decreased anserine levels in the kidney. Both carnosine and lisinopril exert distinct beneficial effects in a standard model of diabetic nephropathy. Both drugs administered together combine the respective effects of single treatment, albeit without exerting additive nephroprotection.

[Modern imaging of kidney tumors]. / Moderne Bildgebung von Nierentumoren.

If a renal mass is suspected on clinical examination or ultrasound the finding has to be confirmed by cross-sectional imaging. Methods that are used include multidetector-row computed tomography (MDCT) and magnetic resonance imaging (MRI). Also contrast-enhanced ultrasound has been successfully implemented in renal imaging and now plays a major role in the differentiation of benign from malignant renal masses. In expert hands it can be used to show very faint vascularization and subtle enhancement. The MDCT technique benefits from the recently introduced dual energy technology that allows superior characterization of renal masses in a single-phase examination, thereby greatly reducing radiation exposure. For young patients and persons allergic to iodine MRI should be used and it provides excellent soft tissue contrast and visualizes contrast enhancement kinetics in multiphase examinations.This article aims at giving a comprehensive overview of these different imaging modalities, their clinical indications and contraindications, as well as a description of imaging findings of various renal masses.

Selection and characterisation of recombinant single-chain antibodies to the hapten Aflatoxin-B1 from naive recombinant antibody libraries.

Selection of antibodies from large repertoire phage display libraries has become a common technique for isolation of specific antibodies to antigens. Many of these libraries are shown to contain antibodies specific to haptens, but only when these haptens are derivatised or conjugated to an immobilising molecule, such as bovine serum albumin (BSA). There has been little demonstration of the suitability of naive recombinant antibody libraries for isolating antibodies that bind low molecular weight haptens in the absence of a carrier molecule and few have addressed the problems associated with selecting antibodies that only recognize the combination of hapten and the carrier molecule. We have panned two-phage antibody libraries against AflatoxinB1-BSA and screened single-chain antibody fragments for binding to AflatoxinB1-BSA and Aflatoxin-B1. Many of the antibodies isolated specifically bound AflatoxinB1-BSA, but not soluble Aflatoxin-B1 or BSA. Modification of the protocol led to isolation of single-chain fragment variable antibody domain (scFv) antibodies that specifically bound soluble Aflatoxin-B1 with an affinity of 6x10(-9) M.

Selection of an anti-CD20, single-chain antibody by phage ELISA on fixed cells.

Cloning the correct genes that code for antibody-variable domains from hybridomas is often complicated by the presence of several immunoglobulin transcripts, some of them arising from a myeloma cell line. For the rapid functional evaluation of recombinant antibody fragments against cell-surface antigens, we established an efficient expression and screening system using phagemid antibodies and fixed cells. VL and VH-polymerase chain reaction (PCR) products, amplified from hybridoma cDNA, were cloned into the phagemid vector pSEX81. After transduction into E. coli and phage rescue, clones were tested for antigen binding using a phage-enzyme-linked immunosorbent assay (ELISA) procedure with whole cells fixed to ELISA wells. This procedure facilitated the successful cloning of a functional anti-CD20, single-chain antibody from hybridoma cDNA. The CD20 B-lymphocyte surface antigen expressed by B-cell lymphomas is an attractive target for cancer treatment using immunoconjugates or bi-specific antibodies.

Generation of a large complex antibody library from multiple donors.

We have generated a large complex library of single chain antibodies based on four individual libraries from each of 50 donors. DNA coding for the heavy and light chain variable domains of the IgM and IgG repertoires was amplified by PCR using two different sets of primers. Each individual library was composed of approximately 1-5x10(7) independent clones giving a final combined library of 4x10(9) members. Screening this library by phage display of single chain antibodies with small haptens, peptides and proteins yielded specific antibodies for each class of antigen.

Screening of phage-displayed antibody libraries.

The problem of amplifying a specific antibody in a population of millions of other antibodies has been solved by the immune system using the process of clonal selection Binding of an antigen to an IgM receptor on the surface of B-lymphocytes stimulates the proliferation and differentiation of the lymphocyte until it matures to an IgG-producing plasma cell. To mimic the first step of this process in bacteria, vectors have been constructed for the expression of antibodies on the surface of bacteria and phages (for review see Chapter 32 ).

Antibodies to steroids from a small human naive IgM library.

Human antibodies specific for digoxigenin, estradiol, testosterone and progesterone have been isolated from a small combinatorial IgM repertoire (4 x 10(7)) of single chain antibodies (scFv). The affinities of both the anti-estradiol and antiprogesterone scFv were approximately 10(8) M(-1). Naive IgM genes appeared to be highly represented, since only the heavy chain variable domain of the anti estradiol antibody contained differences to corresponding germline sequences. The light chain variable domain of the progesterone receptor was also identical to a germline sequence, showing that it is possible for completely naive antibodies to bind steroids with affinities comparable to those obtained after a secondary immune response.

Construction of a semisynthetic antibody library using trinucleotide oligos.

A semisynthetic antibody library composed of single chain Fv fragments (scFv) was constructed by replacing the heavy chain CDR3 region of a human scFv by a random sequence of eight amino acids using trinucleotide codons. After cloning into a phage display vector, an antibody library was generated with a complexity of 8 x 10(8) independent clones. The library was screened for binders to dinitrophenol, fluorescein isothiocyanate and 3-nitro-4-hydroxy-5-iodophenylacetic acid. scFv antibodies that specifically bound the antigen were obtained in each case.

Human antibody libraries in Escherichia coli.

A potentially vast pool of human antibodies with novel specificities for diagnostic and therapeutic purposes can be generated in Escherichia coli. Antibodies to infectious agents have already been isolated by amplifying the heavy and light chain repertoires of donor lymphocytes and they have even been rescued many years after the initial infection from memory cells cultivated in SCID mice. Eventually, however, the creation of extremely large and diverse libraries from the naive antibody repertoire of unactivated B lymphocytes or by gene synthesis using random oligonucleotides for the hypervariable regions could provide a rapid means of obtaining human antibodies to any particular antigen. An important breakthrough for exploiting the potential size and diversity of these libraries has been the development of systems for the surface display of antibodies that are physically linked to their own genes. This allows large numbers of clones to be screened simultaneously and antibodies with affinities of up to 10(8) M-1 have already been obtained using these vectors. It seems quite feasible, therefore, that antibodies with affinities approaching those obtained in the secondary immune response can be obtained by systematically optimizing the strategies for making antibody libraries. Furthermore, it might be possible to establish extremely large antibody repertoires in E. coli by the in vivo recombination of phage and plasmid antibody libraries. The affinity of the selected antibodies could be increased by chain shuffling or random mutagenesis followed by several rounds of selection under increasingly stringent conditions.

Simultaneous mutagenesis of antibody CDR regions by overlap extension and PCR.

A method for the facile simultaneous mutagenesis of complementary-determining regions (CDRs) in a single chain antibody (scFv) is described. Overlapping sets of oligonucleotides containing random sequences within the CDRs corresponding to the heavy chain variable region (VH) jointed to a linker peptide (J) and the light chain variable region (VL) were extended under PCR conditions to full-length genes. These gene products were then further amplified using short PCR primers containing complementary overlaps between the 3' and 5' ends of the VH-J and VL genes respectively. In a final step, the VH-J and VL gene products were mixed and assembled into scFv DNA products by overlap extension under standard PCR conditions. Sequence analyses indicated that the method is basically successful. However, some deletions were observed, which probably reflects difficulties in the automatic synthesis of long degenerate oligonucleotides.

A family of vectors for surface display and production of antibodies.

Expression vectors for surface display and production of single-chain (Fv) antibodies (scAb) have been constructed based on the phagemid pSEX, which expresses DNA encoding a scAb fused to the gene III product of filamentous phage [Breitling et al., Gene 104 (1991) 147-153]. A smaller version of this phagemid, pSEX20, was made by removing an unnecessary cat. To produce a vector for the surface display of other proteins and peptides, the scAb of pSEX20 was substituted by a polycloning site (MCS) to give pSEX40. For the presentation of Ab on the surface of Escherichia coli, phagemid pAP10 was derived from pSEX20 by substituting gene III with a gene encoding the peptidoglycan-associated lipoprotein (PAL). Vectors for producing scAb that can be purified by antibody and metal affinity chromatography were constructed by substituting gene III in the vector pSEX20 with DNA encoding a peptide with a C-terminal epitope recognised by a monoclonal antibody (phagemid pOPE40) or with five C-terminal histidines (pOPE 90).

Recombinant human monoclonal antibodies. Basic principles of the immune system transferred to E. coli.

To produce human monoclonal antibodies in bacteria, a gene repertoire of IgM variable regions was isolated from human peripheral B lymphocytes by the polymerase chain reaction. Alternatively, synthetic antibody genes with random hypervariable regions are being generated that may provide libraries of even higher complexity. For the selection of specific monoclonal antibodies from these libraries, we have developed two E. coli vector systems that facilitate the surface display of an antibody physically linked to its own gene. The phagemid pSEX encodes a fusion protein of an antigen binding domain (Fv-antibody) with the docking protein (pIII) of filamentous phages. Specific antibody genes can therefore be enriched by antigen affinity chromatography. The plasmid pAP1 encodes a fusion protein of an Fv-antibody with a bacterial cell-wall protein. Bacteria carrying this plasmid express functional Fv-antibodies tightly bound to their surface. This should enable the selection of single cells with a fluorescence-assisted cell sorter (FACS) using labeled antigen or by adsorption to immobilized antigen. These vectors permit three major principles of the antibody response to be mimicked in E. coli: 1. Generation of a highly complex antibody repertoire; 2. Clonal selection procedures for library screening; and 3. The possibility of increasing a given affinity by repeated rounds of mutation and selection.