An integrated mechanical degradation model to explore the mechanical response of a bioresorbable polymeric scaffold.

Simulation of bioresorbable medical devices is hindered by the limitations of current material models. Useful simulations require that both the short- and long-term response must be considered; existing models are not physically-based and provide limited insight to guide performance improvements. This study presents an integrated degradation framework which couples a physically-based degradation model, which predicts changes in both crystallinity (Xc) and molecular weight (Mn), with the results of a micromechanical model, which predicts the effective properties of the semicrystalline polymer. This degradation framework is used to simulate the deployment of a bioresorbable PLLA (Poly (L-lactide) stent into a mock vessel and the subsequent mechanical response during degradation under different diffusion boundary conditions representing neointimal growth. A workflow is established in a commercial finite element code that couples both the immediate and long-term responses. Clinically relevant lumen loss is reported and used to compare different responses and the effect of neo-intimal tissue regrowth post-implantation on degradation and on the mechanical response is assessed. In addition, the effects of possible changes in Xc, which could occur during processing and stent deployment, are explored.

Multiscale modeling of trabecular bone marrow: understanding the micromechanical environment of mesenchymal stem cells during osteoporosis.

Mechanical loading directs the differentiation of mesenchymal stem cells (MSCs) in vitro and it has been hypothesized that the mechanical environment plays a role in directing the cellular fate of MSCs in vivo. However, the complex multicellular composition of trabecular bone marrow means that the precise nature of mechanical stimulation that MSCs experience in their native environment is not fully understood. In this study, we developed a multiscale model that discretely represents the cellular constituents of trabecular bone marrow and applied this model to characterize mechanical stimulation of MCSs in vivo.We predicted that cell-level strains in certain locations of the trabecular marrow microenvironment were greater in magnitude (maximum e12»24,000 le) than levels that have been found to result in osteogenic differentiation of MSCs in vitro (>8000 le),which may indicate that the native mechanical environment of MSCs could direct cellular fate in vivo. The results also showed that cell­cell adhesions could play an important role in mediating mechanical stimulation within the MSC population in vivo. The model was applied to investigate how changes that occur during osteoporosis affected mechanical stimulation in the cellular microenvironment of trabecular bone marrow. Specifically,a reduced bone volume (BV) resulted in an overall increase in bone deformation, leading to greater cell-level mechanical stimulation in trabecular bone marrow (maximume12»48,000 le). An increased marrow adipocyte content resulted in slightly lower levels of stimulation within the adjacent cell population due to a shielding effect caused by the more compliant behavior of adipocytes (maximum e12»41,000 le). Despite this reduction, stimulation levels in trabecular bone marrow during osteoporosis remained much higher than those predicted to occur under healthy conditions. It was found that compensatory mechanobiological responses that occur during osteoporosis, such as increased trabecular stiffness and axial alignment of trabeculae, would be effective in returning MSC stimulation in trabecular marrow to normal levels. These results have provided novel insight into the mechanical stimulation of the trabecular marrow MSC population in both healthy and osteoporotic bone, and could inform the design three dimensional(3D) in vitro bioreactor strategies techniques, which seek to emulate physiological conditions.

Bone cell mechanosensation of fluid flow stimulation: a fluid-structure interaction model characterising the role integrin attachments and primary cilia.

Load-induced fluid flow acts as an important biophysical signal for bone cell mechanotransduction in vivo, where the mechanical environment is thought to be monitored by integrin and primary cilia mechanoreceptors on the cell body. However, precisely how integrin- and primary cilia-based mechanosensors interact with the surrounding fluid flow stimulus and ultimately contribute to the biochemical response of bone cells within either the in vitro or in vivo environment remains poorly understood. In this study, we developed fluid-structure interaction models to characterise the deformation of integrin- and primary cilia-based mechanosensors in bone cells under fluid flow stimulation. Under in vitro fluid flow stimulation, these models predicted that integrin attachments on the cell-substrate interface were highly stimulated ε(eq) > 200,000 µÎµ, while the presence of a primary cilium on the cell also resulted in significant strain amplifications, arising at the ciliary base. As such, these mechanosensors likely play a role in mediating bone mechanotransduction in vitro. Under in vivo fluid flow stimulation, integrin attachments along the canalicular wall were highly stimulated and likely play a role in mediating cellular responses in vivo. The role of the primary cilium as a flow sensor in vivo depended upon its configuration within the lacunar cavity. Specifically, our results showed that a short free-standing primary cilium could not effectively fulfil a flow sensing role in vivo. However, a primary cilium that discretely attaches the lacunar wall can be highly stimulated, due to hydrodynamic pressure in the lacunocanalicular system and, as such, could play a role in mediating bone mechanotransduction in vivo.

Cell morphology and focal adhesion location alters internal cell stress.

Extracellular mechanical cues have been shown to have a profound effect on osteogenic cell behaviour. However, it is not known precisely how these cues alter intracellular mechanics to initiate changes in cell behaviour. In this study, a combination of in vitro culture of MC3T3-E1 cells and finite-element modelling was used to investigate the effects of passive differences in substrate stiffness on intracellular mechanics. Cells on collagen-based substrates were classified based on the presence of cell processes and the dimensions of various cellular features were quantified. Focal adhesion (FA) density was quantified from immunohistochemical staining, while cell and substrate stiffnesses were measured using a live-cell atomic force microscope. Computational models of cell morphologies were developed using an applied contraction of the cell body to simulate active cell contraction. The results showed that FA density is directly related to cell morphology, while the effect of substrate stiffness on internal cell tension was modulated by both cell morphology and FA density, as investigated by varying the number of adhesion sites present in each morphological model. We propose that the cells desire to achieve a homeostatic stress state may play a role in osteogenic cell differentiation in response to extracellular mechanical cues.

A fluid-structure interaction model to characterize bone cell stimulation in parallel-plate flow chamber systems.

Bone continuously adapts its internal structure to accommodate the functional demands of its mechanical environment and strain-induced flow of interstitial fluid is believed to be the primary mediator of mechanical stimuli to bone cells in vivo. In vitro investigations have shown that bone cells produce important biochemical signals in response to fluid flow applied using parallel-plate flow chamber (PPFC) systems. However, the exact mechanical stimulus experienced by the cells within these systems remains unclear. To fully understand this behaviour represents a most challenging multi-physics problem involving the interaction between deformable cellular structures and adjacent fluid flows. In this study, we use a fluid-structure interaction computational approach to investigate the nature of the mechanical stimulus being applied to a single osteoblast cell under fluid flow within a PPFC system. The analysis decouples the contribution of pressure and shear stress on cellular deformation and for the first time highlights that cell strain under flow is dominated by the pressure in the PPFC system rather than the applied shear stress. Furthermore, it was found that strains imparted on the cell membrane were relatively low whereas significant strain amplification occurred at the cell-substrate interface. These results suggest that strain transfer through focal attachments at the base of the cell are the primary mediators of mechanical signals to the cell under flow in a PPFC system. Such information is vital in order to correctly interpret biological responses of bone cells under in vitro stimulation and elucidate the mechanisms associated with mechanotransduction in vivo.

A three-scale finite element investigation into the effects of tissue mineralisation and lamellar organisation in human cortical and trabecular bone.

Bone is an exceptional material that is lightweight for efficient movement but also exhibits excellent strength and stiffness imparted by a composite material of organic proteins and mineral crystals that are intricately organised on many scales. Experimental and computational studies have sought to understand the role of bone composition and organisation in regulating the biomechanical behaviour of bone. However, due to the complex hierarchical arrangement of the constituent materials, the reported experimental values for the elastic modulus of trabecular and cortical tissue have conflicted greatly. Furthermore, finite element studies of bone have largely made the simplifying assumption that material behaviour was homogeneous or that tissue variability only occurred at the microscale, based on grey values from micro-CT scans. Thus, it remains that the precise role of nanoscale tissue constituents and microscale tissue organisation is not fully understood and more importantly that these have never been incorporated together to predict bone fracture or implant outcome in a multiscale finite element framework. In this paper, a three-scale finite element homogenisation scheme is presented which enables the prediction of homogenised effective properties of tissue level bone from its fundamental nanoscale constituents of hydroxyapatite mineral crystals and organic collagen proteins. Two independent homogenisation steps are performed on representative volume elements which describe the local morphological arrangement of both the nanostructural and microstructural levels. This three-scale homogenisation scheme predicts differences in the tissue level properties of bone as a function of mineral volume fraction, mineral aspect ratio and lamellar orientation. These parameters were chosen to lie within normal tissue ranges derived from experimental studies, and it was found that the predicted stiffness properties at the lamellar level correlate well with experimental nanoindentation results from cortical and trabecular bone. Furthermore, these studies show variations in mineral volume fraction, mineral crystal size and lamellar orientation could be responsible for previous discrepancies in experimental reports of tissue moduli. We propose that this novel multiscale modelling approach can provide a more accurate description of bone tissue properties in continuum/organ level finite element models by incorporating information regarding tissue structure and composition from advanced imaging techniques. This approach could thereby provide a preclinical tool to predict bone mechanics following prosthetic implantation or bone fracture during disease.

Validation of glutathione quantitation from STEAM spectra against edited 1H NMR spectroscopy at 4T: application to schizophrenia.

OBJECTIVE: Quantitation of glutathione (GSH) in the human brain in vivo using short echo time 1H NMR spectroscopy is challenging because GSH resonances are not easily resolved. The main objective of this study was to validate such quantitation in a clinically relevant population using the resolved GSH resonances provided by edited spectroscopy. A secondary objective was to compare several of the neurochemical concentrations quantified along with GSH using LCModel analysis of short echo time spectra in schizophrenia versus control. MATERIALS AND METHODS: GSH was quantified at 4T from short echo STEAM spectra and MEGA-PRESS edited spectra from identical volumes of interest (anterior cingulate) in ten volunteers. Neurochemical profiles were quantified in nine controls and 13 medicated schizophrenic patients. RESULTS: GSH concentrations as quantified using STEAM, 1.6 +/- 0.4 micromol/g (mean +/- SD, n = 10), were within error of those quantified using edited spectra, 1.4 +/- 0.4 micromol/g, and were not different (p = 0.4). None of the neurochemical measurements reached sufficient statistical power to detect differences smaller than 10% in schizophrenia versus control. As such, no differences were observed. CONCLUSIONS: Human brain GSH concentrations can be quantified in a clinical setting using short-echo time STEAM spectra at 4T.

Isolation and tissue profiles of a large panel of phage antibodies binding to the human adipocyte cell surface.

Phage display is a powerful technique for the rapid selection and isolation of antibodies to any given target antigen. We have applied this technology to isolate over 100 different human antibodies that bind to antigens expressed in situ on the human adipocyte cell surface. This is a diverse panel of antibodies, as indicated by the V-region sequences. The binding profile of each anti-adipocyte antibody has been characterised using phage antibody immunocytochemistry against a panel of normal human tissues. Although there was some variation in the intensity of the adipocyte staining, each antibody consistently recognised adipocytes, where present, irrespective of the tissue source. In addition, all of the antibodies recognised at least one other cell type other than the adipocyte cell surface. In total, over 50 different tissue-binding profiles were recorded, with the most frequently recognised tissues identified as capillaries or smooth muscle. Extensive tissue binding profiles were generated for some antibodies using a panel of 37 different human tissues. This identified anti-adipocyte antibodies with unexpected profiles, such as FAT.13, which binds only to adipocytes and capillaries in the entire tissue panel. We believe this is the most extensive survey ever undertaken of the human adipocyte cell surface. Moreover, similar methodology could be used to derive complete tissue-binding profiles of antibodies against cell-surface antigens of any cell type. Indeed, by screening antibodies on both normal and diseased tissues, it may be possible to identify antigenic associations between different cell types and the pathologies of many diseases.

Tagging bloodmeals with phagemids allows feeding of multiple-sample arrays to single cages of mosquitoes (Diptera: Culicidae) and the recovery of single recombinant antibody fragment genes from individual insects.

A recombinant single-chain variable-region human antibody fragment (scFv) was expressed in Escherichia coli, extracted in hypertonic sucrose, mixed directly with blood and fed to Anopheles gambiae Giles mosquitoes. When E. coli containing the phagemids that encode these scFv were included in bloodmeals, phagemids could be recovered from the mosquito midgut for up to 3 d after feeding. Furthermore, large arrays of such gene-tagged scFv-containing bloodmeals could be fed to cages of mosquitoes using microtiter plates. Arrays of phagemids with and without an antibody insert were fed to single cages of mosquitoes to test whether individual mosquitoes fed from single wells of such arrays. Phagemids were recovered from 95% of blood-fed females and > 80% of these phagemids were monoclonal. Therefore, it is possible to feed multiple sample arrays of recombinant proteins to single cages of mosquitoes and to recover the genetic material that encodes for only one of the array elements from individual mosquitoes. This demonstration indicates that multiple-sample feeding and recovery strategies are feasible and may represent a viable strategy for future rapid screening of biologically active genes, gene products or microorganisms in live arthropods.

Neutralizing human anti crotoxin scFv isolated from a nonimmunized phage library.

Combinatorial phage display technology offers a new possibility for making human antibodies which could be used in immune therapy. We explored the use of this technology to make human scFvs specific for crotoxin, the main toxic component of the venom of the South-American rattlesnake Crotalus durissus terrificus. Crotoxin, a phospholipase A2 neurotoxin constituted by the association of two subunits, exerts its lethal action by blocking neuromuscular transmission. This is the first report of human anticrotoxin scFvs (scFv 1, scFv 6 and scFv 8) isolated from a naive library of more than 1010 scFv clones with in vivo neutralizing activity. Nevertheless, differences are observed at the level of biological and immunological effects. Only scFv 8 is able to reduce the myotoxicity induced by crotoxin and scFv 1 is capable of altering the in vitro enzymatic activity of this toxin. All three scFvs recognize a region of one subunit located at the junction with the other one. Moreover these scFvs share strong amino acid homologies at the level of either the heavy or the light chain. Taken together, our results suggest that the use of human anticrotoxin scFvs may lead to a new and less aggressive passive immune therapy against poisoning by the venom of Crotalus durissus terrificus.

Selection of phage antibodies to surface epitopes of Phytophthora infestans.

Antibodies specific for surface-exposed epitopes on germlings of the plant pathogen, Phytophthora infestans, were isolated from a diverse phage library displaying single-chain Fv (scFv) antibody fragments. The library was subpanned against external soluble components released from mycelia, sporangia and germlings and a discrete population of phage antibodies isolated. Binding of monoclonal phage antibodies was demonstrated by enzyme-linked immunosorbent assay (ELISA) and diversity was established by BstNI restriction enzyme digest patterns. Antibodies were subcloned as fusions at the C-terminus of maltose binding protein (MBP) and expressed as soluble proteins in Escherichia coli. These antibody fusion proteins bound to P. infestans germlings and to mycelial homogenates from various Phytophthora species. The binding activities to mycelial homogenates of fungal species not belonging to the order Peronosporales were substantially lower. Several phage-displayed scFvs were used in conjunction with fluorescently labelled antiphage antibody to visualise the distribution of their cognate epitopes on the surface of the germlings. The combination of procedures developed here with Phytophthora demonstrates the potential of phage antibody technology in isolating antibodies to cell surface and external soluble components of pathogens, some of which may play a role in host/pathogen interactions.

A fully human antibody neutralising biologically active human TGFbeta2 for use in therapy.

Phage display provides a methodology for obtaining fully human antibodies directed against human transforming growth factor-beta (TGFbeta) suitable for the treatment of fibrotic disorders. The strategy employed was to isolate a human single chain Fv (scFv) fragment that neutralises human TGFbeta2 from a phage display repertoire, convert it into a human IgG4 and then determine its TGFbeta binding and neutralisation properties and its physical characteristics. Several scFv fragments binding to TGFbeta2 were isolated by panning of an antibody phage display repertoire, and subsequent chain shuffling of the selected V(H) domains with a library of V(L) domains. The three most potent neutralising antibodies were chosen for conversion to IgG4 format. The IgG4 antibodies were ranked for their ability to neutralise TGFbeta2 and the most potent, 6B1 IgG4, was chosen for further characterisation. 6B1 IgG4 has a high affinity for TGFbeta2 with a dissociation constant of 0.89 nM as determined using the BIAcore biosensor and only 9% cross-reactivity with TGFbeta3 (dissociation constant, 10 nM). There was no detectable binding to TGFbeta1. 6B1 IgG4 strongly neutralises (IC50 = 2 nM) the anti-proliferative effect of TGFbeta2 in bioassays using TF1 human erythroleukaemia cells. Similarly, there was strong inhibition of binding of TGFbeta2 to cell surface receptors in a radioreceptor assay using A549 cells. 6B1 IgG4 shows no detectable cross-reactivity with related or unrelated antigens by immunocytochemistry or ELISA. The 6B1 V(L) domain has entirely germline framework regions and the V(H) domain has only three non-germline framework amino acids. This, together with its fully human nature, should minimise any potential immunogenicity of 6B1 IgG4 when used in therapy of fibrotic diseases mediated by TGFbeta2.

Human antibodies by design.

Monoclonal antibodies (Mabs) have long been considered a good class of natural drugs, both because they mimic their natural role in the body and because they have no inherent toxicity. Although rodent Mabs are readily generated, their widespread use as therapeutic agents has been hampered because they are recognized as foreign by the patient. Evidently, clinical Mabs should be as human as possible and results with some of the more recently developed chimerized and humanized Mabs are testimony to this. Mabs that are entirely human are now being produced from phage display and transgenic mice. The first fully human Mabs generated by phage display have already entered clinical trials, and together with recent advances in these technologies, may finally realize the full potential of antibodies.

Pathfinder selection: in situ isolation of novel antibodies.

BACKGROUND: To devise a novel method for targeted recovery of binding molecules from phage libraries. OBJECTIVES: To assess the potential of the novel technique to the selection of human antibodies to specific cell surface antigens in situ, including carcinoembryonic antigen (CEA), E- and P-selectins, and to the selection of novel antibodies which recognize immobilized purified antigen. STUDY DESIGN: Recovery of these antibodies from a naive human scFv library was effected using a 'pathfinder' molecule. Monoclonal and polyclonal antibodies, as well as natural ligands can serve as pathfinders when conjugated directly or indirectly to horseradish peroxidase (HRP). In the presence of biotin tyramine these molecules catalyze biotinylation of phage binding in close proximity to the target antigen, allowing specific recovery of 'tagged' phage from the total population using streptavidin. In this way, phage binding to the target itself, or in its immediate proximity, are selectively recovered. RESULTS: This work demonstrates that an existing binding specificity can be used as a tool to select phage libraries in situ, obviating the need to purify or clone the target. CONCLUSION: The speed and technical simplicity of this method should find a wide range of applications to phage display libraries, and could be applied to the discovery of new receptors and the elucidation of protein-protein interactions.

Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library.

To generate a stable resource from which high affinity human antibodies to any given antigen can be rapidly isolated, functional V-gene segments from 43 non-immunized human donors were used to construct a repertoire of 1.4 x 10(10) single-chain Fv (scFv) fragments displayed on the surface of phage. Fragments were cloned in a phagemid vector, enabling both phage displayed and soluble scFv to be produced without subcloning. A hexahistidine tag has been incorporated to allow rapid purification of scFv by nickel chelate chromatography. This library format reduces the time needed to isolate monoclonal antibody fragments to under two weeks. All of the measured binding affinities show a Kd < 10 nM and off-rates of 10(-3) to 10(-4) s-1, properties usually associated with antibodies from a secondary immune response. The best of these scFvs, an anti-fluorescein antibody (0.3 nM) and an antibody directed against the hapten DTPA (0.8 nM), are the first antibodies with subnanomolar binding affinities to be isolated from a naive library. Antibodies to doxorubicin, which is both immunosuppressive and toxic, as well as a high affinity and high specificity antibody to the steroid hormone oestradiol have been isolated. This work shows that conventional hybridoma technology may be superseded by large phage libraries that are proving to be a stable and reliable source of specific, high affinity human monoclonal antibodies.

An antibody fragment from a phage display library competes for ligand binding to the low density lipoprotein receptor family and inhibits rhinovirus infection.

Recently antibodies with a wide range of binding specificities have been isolated from large repertoires of antibody fragments displayed on filamentous phage, including those that are difficult to raise by immunization. We have used this approach to isolate an antibody fragment against chicken very low density lipoprotein (VLDL) receptor. It binds to the receptor with good affinity (Kaff = 2 x 10(8) M-1) as measured by plasmon surface resonance, and competes for binding of natural ligands (vitellogenin, VLDL, and receptor-associated protein). The antibody also binds to other members of the low density lipoprotein (LDL) receptor family including rat LDL receptor and human and rat low density lipoprotein receptor-related protein (LRP/alpha 2MR), and it competes for binding of receptor-associated protein to LRP/alpha 2MR. Moreover, the antibody fragment inhibits infection of human fibroblasts deficient in LDL-R but expressing LRP/alpha 2MR by human rhinovirus. Binding of the antibody is abolished upon reduction of the receptors and is strictly Ca2+ dependent. The phage antibody thus recognizes the ligand binding site(s) of several members of the LDL receptor family, in contrast to antibodies produced by hybridoma technology.

Activation of AT1 angiotensin receptors induces DNA synthesis in a rat intestinal epithelial (RIE-1) cell line.

Proliferation of the rat intestinal epithelial cell-line, RIE-1, has previously been shown to be stimulated by certain polypeptide growth factors acting via receptors that possess intrinsic tyrosine kinase activity. In this study, we show that the octapeptide hormone angiotensin II (AII), apparently acting through the AT1 G-protein-coupled receptor, is also a mitogen for RIE-1 cells. Maximal stimulation of DNA synthesis and cellular proliferation occurred at an AII concentration of 10-100 nM, with half-maximal stimulation at 1 nM. The mitogenic response to AII was completely inhibited by the AT1 angiotensin-receptor antagonist, DuP753, but not by the AT2-receptor antagonist, PD123319. The early signalling responses activated by AII in RIE-1 cells include increased production of inositol phosphates, a transient increase in the intracellular concentration of free calcium, an activation of protein kinase C, and a rapid change in the pattern of cellular protein-tyrosine phosphorylation. These results implicate an activation of the inositol lipid signalling pathway via the AT1 receptor subtype in the AII-stimulated mitogenic response of this normal epithelial cell line.

Directed expression of simian virus 40 T-antigen in transgenic mice using the epidermal growth factor gene promoter.

A transgenic mouse line (EGF/Tag) has been established in which expression of SV40 T-antigen is directed by a 5.5 kb fragment of the 5'-flanking region of the mouse epidermal growth factor (EGF) gene. Of the two principal sites of EGF expression in mice, submaxillary gland and kidney, T-antigen mRNA and protein were detected in the former but not in the latter tissue of the EGF/Tag animals. T-antigen expression in the submaxillary gland was restricted to the EGF-producing cells of the granular convoluted tubules, and the oncoprotein induced hyperplasia of these cells. T-antigen levels were markedly higher in the submaxillary glands of male compared with female transgenic mice, suggesting that expression of the transgene was androgen-regulated, like the endogenous EGF gene. These results indicate that the 5.5 kb fragment upstream of the mouse EGF gene contains the DNA enhancer elements required for hormonally regulated expression in the submaxillary gland. Since the hyperplastic submaxillary glands of the EGF/Tag mice continue to synthesize EGF, these glands provide a tissue source from which it may prove possible to establish EGF-secreting cell lines for further in vitro studies of the mechanisms regulating expression of the EGF gene.

Expression of the genes for the epidermal growth factor receptor and its ligands in porcine oviduct and endometrium.

We have used the reverse transcription-polymerase chain reaction (RT-PCR) to determine whether transcripts for the epidermal growth factor (EGF) receptor and its four known ligands--EGF, transforming growth factor alpha (TGF alpha), amphiregulin (Ar), and heparin-binding EGF (HB-EGF)--are expressed in porcine oviduct and endometrium. We were able to detect mRNA for the EGF receptor, EGF, TGF alpha, and Ar in both the oviduct and endometrium, whereas HB-EGF mRNA was not detectable in either tissue. Through use of an antiserum raised against recombinant pig EGF, expression of EGF was found to be localized to the columnar epithelial cells of the oviduct and to the glandular epithelial cells of the endometrium. The possible physiological roles of the EGF family in the reproductive tract are discussed.

Molecular cloning and tissue distribution of pig transforming growth factor alpha.

Transforming growth factor alpha (TGF alpha) was originally identified as a product of tumour tissues and transformed cells in culture. Although it is now clear that expression of this factor is not restricted to neoplastic cells, there remains relatively little information about the sites of expression of TGF alpha in normal tissues. Therefore, an amplified DNA fragment encoding the pig TGF alpha precursor was cloned by reverse transcription-PCR (RT-PCR) using RNA isolated from normal skin tissue as the template. Nucleotide sequence analysis predicts a 160-residue transmembrane polypeptide that differs from the rat, mouse and human TGF alpha precursors at 14, 15 and six sites respectively. The distribution of TGF alpha mRNA in a wide variety of pig tissues was analysed by RT-PCR, using oligonucleotide primers based on the pig TGF alpha cDNA sequence. TGF alpha transcripts were detected in RNA isolated from 17 of the 22 tissues analysed, including four previously unreported sites. Using an antibody raised against a synthetic TGF alpha peptide, we have immunolocalized TGF alpha protein to cells within the red pulp of the spleen and to the distal convoluted tubules of the kidney.