Internal anal sphincter nerves - a macroanatomical and microscopic description of the extrinsic autonomic nerve supply of the internal anal sphincter.

AIM: The internal anal sphincter (IAS) contributes substantially to anorectal functions. While its autonomic nerve supply has been studied at the microscopic level, little information is available concerning the macroscopic topography of extrinsic nerve fibres. This study was designed to identify neural connections between the pelvic plexus and the IAS, provide a detailed topographical description, and give histological proof of autonomic nerve tissue. METHODS: Macroscopic dissection of pelvic autonomic nerves was performed under magnification in seven (five male, two female) hemipelvises obtained from body donors (67-92 years). Candidate structures were investigated by histological and immunohistochemical staining protocols to visualize nerve tissue. RESULTS: Nerve fibres could be traced from the anteroinferior edge of the pelvic plexus to the anorectal junction running along the neurovascular bundle anterolaterally to the rectum and posterolaterally to the prostate/vagina. Nerve fibres penetrated the longitudinal rectal muscle layer just above the fusion with the levator ani muscle (conjoint longitudinal muscle) and entered the intersphincteric space to reach the IAS. Histological and immunohistochemical findings confirmed the presence of nerve tissue. CONCLUSIONS: Autonomic nerve fibres supplying the IAS emerge from the pelvic plexus and are distinct to nerves entering the rectum via the lateral pedicles. Thus, they should be classified as IAS nerves. The identification and precise topographical location described provides a basis for nerve-sparing rectal resection procedures and helps to prevent postoperative functional anorectal disorders.

The use of a cocktail of single chain Fv antibody fragments to improve the in vitro and in vivo targeting of melanoma.

Radioscintigraphy using single chain antibody fragments (scFvs) offers a potential means of early detection of melanoma metastases. However, previous studies have shown suboptimal levels of tumour localization and non-specific background accumulation which may be due to antigen heterogeneity. We aimed to improve tumour localization by using a cocktail of different scFvs targeting different epitopes on melanoma cells. We have previously developed three scFvs against distinct and highly tumour-specific melanoma cell-surface antigens by chain shuffling and antibody phage selection on melanoma cells. Three scFvs, RAFT3, B3 and B4 were labeled with 125Iodine and tested both individually and as a cocktail in a nude mouse xenograft model for human melanoma. Results demonstrated improved tumour localization in vivo when compared to the individual scFvs. Tumour uptake of the cocktail at 1 hour was 24.220% ID/g (injected dose/gram) compared with 2.854%, 2.263% and 1.355% for B4, RAFT3 and B3, respectively, when injected individually. In addition, the cocktail exhibited significantly superior tumour to normal tissue ratios for muscle and spleen (p<0.05). A combination or 'cocktail' of scFv clones may have an advantage over individual scFvs for melanoma targeting in patients because of heterogeneity in the expression of different epitopes of antigens on melanoma cells.

Reducing renal accumulation of single-chain Fv against melanoma-associated proteoglycan by coadministration of L-lysine.

In view of the rising melanoma incidence and the absence of effective treatments for metastatic disease, there is an urgent need for new methods that allow the early detection of melanoma. To this end, in vivo detection by patient imaging with single-chain Fv (scFv) antibody fragments is an attractive diagnostic approach. However, high non-specific accumulation of scFvs in the kidney reduces image quality in this body area and prevents the use of scFvs for melanoma radioimmunotherapy. We have tested the effect of coadministration of L-lysine with (125)I-labelled scFvs against melanoma-associated proteoglycan on kidney accumulation in a nude mouse xenograft model. Coadministration of L-lysine had no significant effect on tumour accumulation of scFvs or blood clearance, but decreased kidney accumulation by factors of 2.25, 2.3, 6.3 and 5.8, respectively, at 1, 3, 6 and 18 h post-injection, and improved tumour to muscle contrast. The reduction in kidney accumulation was maximal at time points that can be extrapolated to patient studies. The time dependence of the effect suggests that further improvements could be achieved with an optimized dosing regimen. When combined with other strategies to reduce kidney accumulation of scFvs, coadministration of L-lysine has the potential to significantly improve tumour to kidney contrast.

Improved production by domain inversion of single-chain Fv antibody fragment against high molecular weight proteoglycan for the radioimmunotargeting of melanoma.

Melanoma is among the few cancers with rising incidence. Currently there is no effective treatment for metastatic disease, but improved detection of melanoma has the potential to benefit the management of patients with early disease. Radioimmunodection by imaging with single-chain Fv (scFv) antibody fragments is one such emerging diagnostic method. However, the amount of scFv that can be produced at a scale suitable for use in patients is limiting. We have previously shown that the bacterial expression of a scFv derived from a monoclonal antibody (MAb) specific for melanoma-associated proteoglycan can be increased by light chain shuffling. In this report we show that a further increase in expression yield can be obtained by reversing the usual V(H)-V(L) orientation of scFvs to V(L)-V(H). Such seemingly minor changes have previously been reported to have unexpected effects on the in vitro and in vivo binding properties of recombinant antibodies. Our results show that reversal of the V domain orientation of the scFv improves expression by 150% without an adverse effect on melanoma binding in vitro and tumor targeting in vivo. Therefore, our results show that alteration of V domain orientation can improve the production yield of clinically useful antibody fragments. When used in combination with other antibody engineering approaches for increased antibody production changing the domain orientation is a simple strategy to achieve significant improvements in the production of scFvs for tumor radioimmunodetection for patient studies.

In vivo targeting of malignant melanoma by 125Iodine- and 99mTechnetium-labeled single-chain Fv fragments against high molecular weight melanoma-associated antigen.

Monoclonal antibodies (MAbs) against high-molecular-weight melanoma-associated antigen (HMW-MAA) have been used in vivo to target melanoma. More recently, single chain Fv (scFv) antibody fragments against HMW-MAA have been described that may improve melanoma targeting. However, there have been few in vivo studies with antimelanoma scFvs because these have proved difficult to label with isotopes (e.g., 99mTc) suitable for imaging. We have generated a series of scFvs against HMW-MAA by chain shuffling and antibody phage selection on melanoma cells. In preliminary experiments we identified one scFv (RAFT3) as suitable for in vivo melanoma targeting. Direct radiolabeling of RAFT3 scFv with 99mTc was simple, yielding a radiochemical purity of >90%. The label remained stable for 24 h in vitro. 125I- and 99mTc-labeled RAFT3 scFv were tested in a nude mouse xenograft model for human melanoma and were compared with the parent MAb LHM2 and its F(ab')2 fragment versus nonmelanoma-specific MAb and scFv. RAFT3 scFv accumulated specifically in the tumor and showed greater tumor specificity compared with LHM2 with faster pharmacokinetics (t(1/2)alpha, 8 min; t(1/2)beta, 189 min; and t(1/2)alpha, 37 min; t(1/2)beta, 384 min, respectively) and reduced background in liver, lung, and spleen. Nonspecific accumulation of 99mTc-labeled RAFT3 scFv in the kidney was high but tumor:normal tissue ratios were better compared with 125I-labeled RAFT3 scFv and LHM2 F(ab')2. Overall, tumor-targeting efficiency at equivalent time points was scFv > IgG > F(ab')2 in good agreement with previously described scFvs engineered for 99mTc labeling. We discuss the potential use of RAFT3 scFv for imaging and therapy of metastatic melanoma.

Retrovirus targeting by tropism restriction to melanoma cells.

Targeted vectors will be necessary for many gene therapy applications. To target retroviruses to melanomas, we fused a single-chain variable fragment antibody (scFv) directed against the surface glycoprotein high-molecular-weight melanoma-associated antigen (HMW-MAA) to the amphotropic murine leukemia virus envelope. A proline-rich hinge and matrix metalloprotease (MMP) cleavage site linked the two proteins. The modified viruses bound only to HMW-MAA-expressing cells, as inclusion of the proline-rich hinge prevented viral binding to the amphotropic viral receptor. Following attachment to HMW-MAA, MMP cleavage of the envelope at the melanoma cell surface removed the scFv and proline-rich hinge, allowing infection. Complexing of targeted retroviruses with 2, 3-dioleoyloxy-N-[2(spermine-carboxamido)ethyl]N, N-dimethyl-1-propanaminium trifluoroacetate-dioleoyl phosphatidylethanolamine liposomes greatly increased their efficiency without affecting their target cell specificity. In a cell mixture, 40% of HMW-MAA-positive cells but less than 0.01% of HMW-MAA-negative cells were infected. This approach can therefore produce efficient, targeted retroviruses suitable for in vivo gene delivery and should allow specific gene delivery to many human cell types by inclusion of different scFv and protease combinations.

Isolation of human tumor-specific antibodies by selection of an antibody phage library on melanoma cells.

A human single-chain Fv (scFv) library as fusion to phage was constructed from donors with a high titer of autoantibodies. The library was subjected to three rounds of positive selection on human melanoma cells and negative selection on human peripheral blood mononuclear cells. Two scFv clones, B3 and B4, were isolated that bound melanoma cells in cell ELISA and fluorescence-activated cell sorting. The scFvs were characterized further by immunohistochemistry on a large number of normal human tissues. No cross-reactivity with normal tissues was observed. On the other hand, the target antigens were expressed in sections from several different melanoma patients and in some breast cancer and basal cell carcinoma sections. The unusually high tumor specificity of the B3 and B4 antigens makes them attractive targets for the specific therapy of melanoma. The selection strategy used should be generally applicable to the identification of novel cell surface antigens by antibody phage display.

Efficient in vivo targeting of malignant melanoma by single-chain Fv antibody fragments.

Single-chain antibody fragments (scFvs) have superior pharmacokinetic and biodistribution characteristics compared with larger antibody fragments when used for radioimaging of human tumours. An scFv specific for malignant melanoma (B4 scFv) was tested in a mouse xenograft model for human melanoma and compared with the monoclonal antibody (MAb) LHM2. LHM2 is specific for the high molecular weight proteoglycan melanoma-associated antigen (HMW-MAA), while B4 scFv binds a novel melanoma-specific antigen. The B4 scFv was cleared rapidly from the circulation (t(1/2)alpha = 7 min) compared with LHM2 MAb (t(1/2)alpha = 37 min). However, the B4 scFv had an unusually long t(1/2)beta (437 min compared with 384 min for LHM2 MAb). Biodistribution studies showed that B4 accumulated specifically in the tumour grafts. Although non-specific accumulation in the liver, lung, spleen and blood was lower than with LHM2, non-specific accumulation of B4 in the kidney was higher. Despite this, the overall targeting efficiency in this study, as determined by tumour:normal tissue ratios, showed that B4 was superior to whole antibody. Immunoscintigraphy images showed good correlation with the biodistribution data for B4 and LHM2. This study represents the first use of an anti-melanoma scFv in vivo.

Retroviral vector targeting to melanoma cells by single-chain antibody incorporation in envelope.

Two strategies for targeting recombinant retroviruses to melanoma cells were compared. One was to extend the tropism of an ecotropic envelope to human melanoma cells, the other was to enhance the tropism of an amphotropic envelope for melanoma cells. Chimeric retroviral envelopes, incorporating a single-chain antibody (ScFv) directed against high-molecular-weight melanoma-associated antigen (HMWMAA) at the amino terminus are correctly processed and incorporated into virions. ScFv-ecotropic envelope chimeras allow specific, but low-titer, targeting of HMWMAA-positive cells, when co-expressed with ecotropic envelopes. ScFv-amphotropic envelope chimeras bind specifically to HMWMAA-positive cells and allow preferential infection at high titer.

Melanoma antigens and targets for vaccination.

Metastatic melanoma is at present incurable by conventional therapy. However, encouraging improvements in patients survival have been obtained in clinical trials with melanoma vaccines as active specific immunotherapy. This review will discuss recent developments in melanoma vaccines.

Generation and selection of monoclonal antibodies, single-chain Fv and antibody fusion phage specific for human melanoma-associated antigens.

A panel of 13 murine monoclonal antibodies (mAbs) recognizing antigens on human melanoma cells but not on melanocytes was generated. Two mAbs (LHM3 and LHM5) stained sections of melanoma but not normal tissues. mAbs LHM2 and LHM8 stained only a minority of normal tissues. The mAbs differed further in their staining patterns on melanoma cell lines HMB2, DX3 and SK23 in FACS. The mAbs recognize antigens of 34, 38, 57, 94, 190-200 and > 200 kD. One mAb each bound to each of the antigens HLA DR (LHM4) and high molecular weight proteoglycan (LHM2). The high molecular weight proteoglycan-specific mAb was used to construct a single-chain Fv (scFv) antibody fragment and an antibody fusion phage in Escherichia coli. Both the scFv and the fusion phage were shown to bind specifically to melanoma cells. A method for the selection of melanoma cell-binding phages from phage libraries is described.

Expression of murine soluble CD4 protein in baculovirus infected insect cells.

The expression of murine soluble CD4 (L3T4) protein (sCD4) by baculovirus-infected insect cells was characterized. The yield of sCD4 reached 2 mg/l culture supernatant late in infection. Nevertheless, a large amount of sCD4 remained cell-associated, presumably in the endoplasmic reticulum or an early golgi compartment, as indicated by the endo-beta-N-acetyl-D-glucosaminidase H (endo-H) sensitivity of its carbohydrate chains. The secreted form of sCD4 is modified with both endo-beta-N-acetyl-D-glucosaminidase D (endo-D) and endo-H-sensitive oligosaccharides. It was possible that the incomplete secretion indicated faulty glycosylation or improper folding of the sCD4 protein. However, inhibitor studies showed that complete carbohydrate processing is not required for secretion of sCD4 by insect cells. Moreover, maintained reactivity with a panel of monoclonal Ab as well as phase partitioning experiments suggested that secretion is apparently not caused by misfolding of the sCD4 protein. Similar results were obtained with biologically active murine interleukin-4 produced by insect cells. This indicates that an inefficient secretory pathway may be a general problem of baculovirus-infected insect cells and is not a consequence of incorrect molecular conformation.

Biochemical evidence of the physical association of the majority of CD3 delta chains with the accessory/co-receptor molecules CD4 and CD8 on nonactivated T lymphocytes.

The association of components of the CD3 complex with the accessory molecules CD4 and CD8 was studied by immunoprecipitation experiments followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Enhanced surface iodination was achieved by a water-soluble derivative of the Bolton-Hunter reagent. Using freshly isolated nonactivated splenic T cells, we find that antibodies to CD4 and to CD8 strongly co-precipitate a 28-30-kDa band identical in mobility to the delta chain of the CD3 complex. Components corresponding in mobility to the epsilon and gamma chains of the CD3 complex are also co-precipitated but to a much lesser extent. The identity of the co-precipitated 28-30-kDa material with the CD3 delta chain was ascertained by two-dimensional nonreducing/reducing SDS-PAGE, by two-dimensional non-equilibrium pH gradient electrophoresis/SDS-PAGE and by one-dimensional peptide mapping with three different proteases. The co-precipitated 28-30-kDa material was identical to the CD3 delta chain by all these criteria. Quantitative analyses by densitometric gel tracing revealed that the amounts of CD3 delta co-precipitated with anti-CD4 and anti-CD8 add up to those in anti-V beta precipitates and to an average of 90% of those in anti-CD3 epsilon precipitates. We conclude that the majority of CD3 delta chains are associated with the accessory/co-receptor molecules CD4 or CD8 on resting T cells, and that this association is independent of antigen-specific recognition by the T cell receptor.

Affinity enhancement and transmembrane signaling are associated with distinct epitopes on the CD8 alpha beta heterodimer.

CD8 is a heterodimeric membrane glycoprotein on MHC class I-restricted T lymphocytes that cooperates with the alpha beta CD3 TCR in the recognition of MHC class I molecules presenting antigenic peptides. Co-operation has two components: enhancement of the affinity of MHC/peptide-TCR interaction, and signal transduction through the T cell membrane. The cytolytic function of CTL is primarily dependent on the affinity-enhancement component of CD8-TCR cooperation whereas activation of resting CD8+ T cells is primarily dependent on transmembrane signaling. Using a panel of mAb, two to the alpha-chain and three to the beta-chain of CD8, we investigated the relationships between epitopes and functional regions of the CD8 molecule. Two of the antibodies, one to the alpha-chain and one to the beta-chain of CD8, inhibit the cytolytic function of CTL but not the generation of CTL from resting T cells. Another two antibodies, also one to the alpha- and one to the beta-chain, inhibited the generation of CTL while enhancing the cytolytic function of CTL. These results suggest that both the alpha- and beta-chain of CD8 possess two distinct regions, one involved in affinity enhancement and the other in transmembrane signaling. The former may be the MHC class I-binding region whereas the latter may associate with the alpha beta CD3 TCR. The data can explain the apparent functional equivalence of CD8 alpha alpha homodimers and alpha beta heterodimers.

A comparative assessment of the roles of CD8 and CD2 in the functions of activated murine CD8+ T lymphocytes.

Both CD8 and CD2 are T cell surface receptors involved in physical cell interaction and in transmembrane signalling. The present paper addresses their role in the induction of two different functions of the cloned murine cytotoxic T cell C196: target cell lysis and IFN-gamma production. These functions were induced in C196 either by stimulation with the specific stimulator/target cell P815 or, bypassing specific recognition, by the aCD3 hybridoma 145-2C11 or by solid phase aTCR antibodies. These responses were tested for their susceptibility to inhibition/enhancement by a panel of aCD8 and aCD2 mAb. In addition, CD8 deficient and CD8/CD2 double-deficient variants of C196 were transfected with the CD8 and CD2 genes and the resulting cell lines were analysed for their functional capacities. The following results were obtained: (i) CD8 is primarily important in the specific recognition process of activated CTL; (ii) transmembrane signalling of activated CTL through the TCR does not require CD8, nor is it sensitive to modification through CD8; (iii) CTL can nevertheless be directly activated through CD8; however, this is restricted to induction of cytotoxicity but does not result in IFN-gamma production; (iv) CD2 does not seem to be important in any of these responses.

Production of murine interleukin-4 and interleukin-5 by recombinant baculovirus.

The cDNAs coding for murine interleukin-4 and -5 have been expressed using the baculovirus AcNPV as a vector in insect cells. Interleukins are secreted into the culture medium of virus-infected insect cells at high levels. Recombinant baculoviruses were isolated using a simple and fast selection scheme based on assays for interleukin activity. The cloning strategy described should be generally applicable to the production of any interleukin or other proteins with biological activity in the baculovirus system.

Analysis of structural and biological parameters affecting plasmid deletion formation in Bacillus subtilis.

Deletions generated following stimulation by the deletion hot spot of plasmid pHV15-1 were studied in Bacillus subtilis. Nucleotide sequencing showed that deletions extend between short direct repeat sequences. Such direct repeat sequences may have homology to the sequence of the hot spot. Deletion formation is recE-independent, but requires an active exonuclease V (AddAB) enzyme. Other structural parameters like plasmid size and structure influence deletion formation.

A plasmid vector for the isolation of transcriptional terminators in Bacillus subtilis.

A plasmid vector has been constructed that permits the positive selection of Bacillus subtilis transcriptional terminator signals. The usage of the vector and its possible applications to the analysis of gene regulation in B. subtilis are described.

DNA methyltransferase genes of Bacillus subtilis phages: structural relatedness and gene expression.

The DNA methyltransferase (Mtase) genes of temperate Bacillus subtilis phages phi 3T, rho 11 and SP beta were cloned and expressed in Escherichia coli. Each gene specifies a 47-kDa1 protein, which modifies BsuR (GGCC) and Fnu4HI (GCNGC) target sequences. Transcription is controlled by phage promoters located on the cloned fragments. The direction of transcription and the approximate position of the Mtase genes were determined. DNA/DNA hybridization experiments revealed close structural relatedness of the phi 3T, rho 11 and SP beta genes. A significant degree of homology was also found among these genes and the Mtase gene of related phage SPR, which codes for an enzyme with different modification specificity. These results suggest a common ancestor of the different phage Mtase genes. Phage Z, the only BsuR-sensitive member of this phage group, lacks a modification gene, but contains regions homologous to sequences flanking the SPR, phi 3T, rho 11 and SP beta Mtase genes.