Interleukin-10 Gene Transfer in Rat Limbal Transplantation.

PURPOSE: To evaluate the gene transfer of the interleukin (IL)-10 cytokine as a treatment modality for prolonging limbal allograft survival in a rat model. MATERIALS AND METHODS: Adenoviral (AV) and lentiviral (LV) vectors were produced for ex vivo gene transfer into limbal graft tissue prior to orthotopic transplantation. Experimental groups comprised unmodified isografts, unmodified allografts, allografts transfected with a reporter gene, and allografts transfected with IL-10. The functional effects of the transgenes were determined by clinical assessment and by following donor cell survival in the recipient animal. Group comparisons were made using survival analysis and tested with the log-rank test. Differences in mean rejection times between groups were tested using the Wilcoxon rank-sum test. RESULTS: Isografts survived during the entire observation period of 56 days. Allografts underwent clinical rejection at a mean of 6.7 days (standard deviation 2.0) postoperatively, irrespective of the presence of transgenes (p < 0.001 for difference in rejection times). For both the AV and LV vector systems, Kaplan-Meier analysis showed a statistically significant difference with respect to time-to-graft failure when comparing allografts transfected with IL-10 with allografts transfected with reporter gene alone (p = 0.011 and p < 0.001, respectively). In the isografts, donor cells could be detected during the complete observation period. In all the allograft groups, however, donor cell detection declined after 1 week and was lost after 4 weeks. CONCLUSIONS: Under the conditions tested in the present model, both the AV and the LV vector systems were able to transfect limbal graft tissue ex vivo with biologically active IL-10, leading to delayed rejection compared to the controls.

Co-expression of a scFv antibody fragment and a reporter protein using lentiviral shuttle plasmid containing a self-processing furin-2A sequence.

It is often desirable to co-express a reporter protein with a potential therapeutic protein, to verify correct targeting of an expression strategy. Vectors containing a viral self-processing 2A sequence have been reported to drive equimolar expression of two or more transgenes from a single promoter. Here, we report on the co-expression of a secreted antibody fragment and an intracellular reporter protein, enhanced yellow fluorescent protein from lentiviral shuttle plasmids by inserting a furin-2A (F2A) sequence between the two cDNAs, in two different orientations, in the expression cassette. We show that the order of these two transgenes relative to the F2A sequence affects expression levels. Reduced expression of each transgene positioned downstream of F2A, compared with upstream of F2A, was observed (p<0.05). Moreover, protein expression from double-cDNA plasmids was significantly lower than from their corresponding single transgene counterparts (p<0.05).

Distinct microRNA profiles are associated with the severity of hepatitis C virus recurrence and acute cellular rejection after liver transplantation.

Recurrent hepatitis C virus (HCV) infection is associated with accelerated fibrosis rates after liver transplantation (LT) and is the leading cause of graft failure. Furthermore, distinguishing recurrent HCV from acute cellular rejection (ACR) can be problematic, and this can lead to inappropriate treatments and adverse outcomes. We hypothesized that intragraft microRNA (miRNA) expression profiles could distinguish the severity of recurrent HCV and differentiate recurrent HCV from ACR. We established meticulously matched post-LT patient cohorts in order to derive robust global miRNA expression profiles and minimize the impact of variables known to influence HCV recurrence. These cohorts consisted of patients with slow HCV fibrosis progression (Ishak stage < F2), fast HCV fibrosis progression (Ishak stage ≥ F2), ACR, and nonviral etiologies. We found increased intragraft expression of miRNA-146a, miRNA-19a, miRNA-20a, and miRNA-let7e in slow progressors versus fast progressors, and we validated these findings with quantitative PCR. This miRNA network regulates the expression of cardinal genes implicated in promoting antifibrogenic, antiangiogenic, and anti-inflammatory pathways. miRNA-19a and miRNA-20a were also specifically detected in the serum of slow progressors. Furthermore, intragraft miRNA expression distinguished fast HCV progression from ACR. Here, changes in the expression of key miRNAs regulating fibrogenic and angiogenic pathways were associated with fast HCV progression. We demonstrate specific miRNA expression signatures that discriminate the rates of fibrosis progression in patients with recurrent HCV, and we distinguish recurrent HCV from ACR after LT. A pathway analysis indicates that specific miRNAs may play a regulatory role in these processes. Selected miRNAs may serve as intragraft and serum biomarkers for recurrent HCV after LT and help to distinguish between ACR and recurrent HCV.

Stability of small non-coding RNA reference gene expression in the rat retina during exposure to cyclic hyperoxia.

PURPOSE: Oxygen-induced retinopathy (OIR) is a robust animal model of human retinopathy of prematurity that readily allows changes in retinal gene and microRNA (miRNA) expression in response to fluctuations in oxygen levels to be studied. We sought to identify small non-coding RNA (ncRNA) genes that showed stable expression upon exposure to varying levels of oxygen, with different developmental stages and in different rat strains, to act as reference genes for normalizing miRNA expression in a rat model of OIR. METHODS: Expression of five small ncRNAs (U6 snRNA, miR-16, U87, 4.5S RNA (H) "Variant 1", and 5S ribosomal RNA [rRNA]) were tested on a standard RNA pool and representative retinal samples from P5, P6, P9, and P14 from room air- and cyclic hyperoxia-exposed rats using reverse transcription (RT)-qPCR, to assess the effect of developmental stage and exposure to fluctuations in oxygen levels, respectively. Two strains of inbred albino rats, Fischer 344 (F344, resistant to OIR) and Sprague-Dawley rats (SD, susceptible to OIR), were used to assess the effect of rat strain on the stability of the small ncRNAs. RESULTS: In this rat model of OIR, 5S rRNA expression was variable with strain, fluctuations in oxygen levels, and developmental stage. U6 snRNA was stably expressed with changes in oxygen levels, and minimal variation was observed with strain and developmental stage. MiR-16 showed less stable expression with changes in oxygen levels and between strains compared to U6 snRNA. Some variation in expression in response to developmental stage was also observed. The PCR amplification efficiencies of the U6 snRNA and miR-16 TaqMan assays were 56% and 78%, respectively. U87 and 4.5S RNA (H) "Variant 1" expression varied with strain, exposure to cyclic hyperoxia, and in particular developmental stage, and was at low levels in the neonatal rat retina. CONCLUSIONS: We conclude that U6 snRNA and miR-16 are the most suitable reference RNAs for normalizing miRNA expression, as they are relatively stable with strain, exposure to cyclic hyperoxia, and developmental stage in a rat model of OIR.

Expression of an anti-CD4 single-chain antibody fragment from the donor cornea can prolong corneal allograft survival in inbred rats.

AIM: To investigate whether expression of an anti-CD4 antibody fragment (scFv) by a lentivector-transduced donor cornea can prolong rat corneal allograft survival. METHODS: Inbred Fischer 344 rats received penetrating corneal allografts from Wistar-Furth donors after a 3 h transduction of the donor cornea with a lentivector carrying anti-CD4scFv cDNA (Lv-CD4scFv), a lentivector carrying the reporter gene-enhanced yellow fluorescence protein (LV-eYFP), or an adenoviral vector carrying anti-CD4 scFv cDNA (Ad-CD4scFv). Unmodified controls were also performed. Graft survival was assessed by corneal clarity, and rejection was confirmed histologically. RESULTS: In organ-cultured corneas, expression of anti-CD4 scFv was detected at 2 days post-transduction with the adenoviral vector, compared with 5 days post-transduction with the lentivector, and was 10-fold higher than the former. More inflammation was observed in Ad-CD4scFv-modified allografts than in Lv-CD4scFv-modified grafts at 15 days postsurgery (p=0.01). The median time to rejection for unmodified, LV-eYFP and Ad-CD4scFv grafts was day 17, compared with day 22 for Lv-CD4scFv grafts (p≤0.018). CONCLUSION: Donor corneas transduced with a lentiviral vector carrying anti-CD4scFv cDNA showed a modest but significant prolongation in graft survival compared with unmodified, Lv-eYFP and Ad-CD4scFv grafts. However, rejection still occurred in all Lv-CD4scFv grafts, indicating that sensitisation may have been delayed but was not prevented.

The influence of cervical and thoracic lymphadenectomy on corneal allograft rejection in inbred rats.

AIM: To investigate the site of alloantigen presentation in the rat following orthotopic corneal transplantation. METHODS: Adult inbred Fischer 344 rats received penetrating corneal allografts from inbred Wistar Furth donors (n=17), without lymphadenectomy. A second group (n=8) underwent bilateral removal of superficial cervical and facial lymph nodes 7 days before transplantation. A third group (n=9) underwent bilateral removal of superficial cervical, facial, internal jugular and posterior cervical nodes. Graft survival was assessed by corneal clarity and rejection was confirmed histologically. RESULTS: All allografts underwent rejection. The median time to rejection for unmodified allografts was day 15, compared with day 14.5 for minimally lymphadenectomised recipients and day 18 for more extensively lymphadenectomised recipients (p>0.05, all comparisons). The median day to rejection for the combined group of lymphadenectomised rats was day 17 (p>0.05 compared with unmodified grafts). The rejection process was similar in all recipients. CONCLUSIONS: Removal of multiple lymph nodes in the neck and thorax did not significantly influence the incidence, tempo or nature of the corneal allograft response. Sensitisation and clonal expansion of corneal alloantigen-reactive cells cannot occur only in superficial cervical, facial, internal jugular and posterior cervical lymph nodes in the rat.

Lentivirus-mediated gene transfer of interleukin 10 to the ovine and human cornea.

BACKGROUND: Gene transfer to a donor cornea ex vivo can modulate corneal graft failure in experimental animal models. We compared a lentiviral vector (LV) carrying the transgene ovine interleukin 10 (IL10) with a comparable adenoviral vector (Ad) for its ability to transduce ovine and human corneas and to modulate ovine corneal allograft survival. METHODS: The LV carrying the ovine IL10 gene was used to transduce ovine and human corneas in vitro. LV-mediated gene expression in corneal endothelium was assessed by real-time quantitative reverse-transcriptase polymerase chain reaction, at varying doses and duration of transduction. The effect of ex vivo transduction of the donor cornea with LV-SV40-IL10 was assessed following orthotopic corneal transplantation in outbred sheep. RESULTS: Expression of IL10 mRNA in Ad-CMV-IL10-transduced ovine corneas was 10(3)-fold higher than in LV-SV40-IL10-transduced corneas (P < 0.0001), and 10(7)-fold higher than in non-transduced controls. IL10 was secreted rapidly from Ad-CMV-IL10-transduced, organ-cultured corneas, peaking at 13-15 days. IL10 secreted from LV-SV40-IL10-transduced corneas increased 20-fold compared with controls, but had not reached a plateau at 15 days. Gene expression driven by LV-SV40-IL10 varied with vector dose and transduction time, but was less than with Ad-CMV-IL10 at both mRNA and protein levels. Gene expression driven by LV-SV40-IL10 was faster in the human cornea than the ovine cornea. Corneal allograft survival was prolonged by a median of 7 days in the LV-SV40-IL10-treated recipients, compared with the control group (P = 0.026). CONCLUSION: Although lentiviral vectors show some promise for corneal gene therapy, they are less efficient than adenoviral vectors.

Hereditary influences in oxygen-induced retinopathy in the rat.

Rodent models of oxygen-induced retinopathy (OIR) provide important insights into the pathogenesis of human retinopathy of prematurity. Herein, we present an overview of our work with rat OIR to date. We have identified marked and consistent variations in susceptibility to OIR amongst different inbred rat strains and provide strong evidence for a genetic determinant of susceptibility to OIR. Furthermore, we have characterised differences in retinal angiogenic factor gene expression amongst different inbred rat strains exposed to cyclic hyperoxia. A key determinant of susceptibility to OIR appears to be the extent to which pro-angiogenic factor genes, such as vascular endothelial growth factor and erythropoietin, are expressed during the period of hyperoxic exposure. Those strains in which expression is relatively well maintained are less susceptible to retinopathy than are those in which expression is reduced. In addition, we identify an association between ocular pigmentation and OIR susceptibility.

Production of scFv antibody fragments from a hybridoma with functional activity against human vascular endothelial growth factor.

Vascular endothelial growth factor (VEGF) plays a major role in the development of aberrant neovascularization in ocular diseases such as diabetic retinopathy and age-related macular degeneration (ARMD), and is an important therapeutic target for these diseases. Monoclonal antibodies specific for VEGF are in clinical use for some patients with ARMD, delivered by intraocular injection. We have shown previously that single chain antibody fragments (scFv) penetrate into the eye when applied topically to the ocular surface. Here we describe the production of a scFv from a monoclonal antibody specific for human VEGF and demonstrate its ability to decrease proliferation of human umbilical vein endothelial cells in culture. A suitably formulated anti-VEGF scFv may have potential as a less invasive topical treatment for potentially blinding neovascular diseases of the eye.

The potential of viral vector-mediated gene transfer to prolong corneal allograft survival.

The cornea is a particularly attractive target for gene therapy designed to improve the outcome of corneal transplantation. First, there is a clear and well-defined clinical need. Second, because donor corneas can be preserved for days if not weeks within an eye bank, ex vivo transduction of a donor cornea can be carried out without the urgency associated with many other forms of transplantation. Finally, the partial sequestration of the eye from the systemic circulation decreases the likelihood of spillover of vector and transgene, and the immune privileged nature of the cornea and anterior segment affords a degree of protection from immune responses directed against the vector. A wide range of vectors has been investigated for gene transfer to the cornea. A number of viral vectors, in particular, have proved to be efficient at transducing the cornea and in association with a variety of transgenes, have been used successfully to prolong corneal allograft survival significantly in animal models. The most suitable such vector for future clinical studies in corneal transplantation has yet to be determined, but the most likely include recombinant adenoviral, adeno-associated viral and lentiviral vectors. In this review, we examine the ability of these viral vectors to transduce the cornea, and summarise those studies in which gene therapy has been used to prolong experimental corneal allograft survival.

Gene expression microarray analysis of early oxygen-induced retinopathy in the rat.

Different inbred strains of rat differ in their susceptibility to oxygen-induced retinopathy (OIR), an animal model of human retinopathy of prematurity. We examined gene expression in Sprague-Dawley (susceptible) and Fischer 344 (resistant) neonatal rats after 3 days exposure to cyclic hyperoxia or room air, using Affymetrix rat Genearrays. False discovery rate analysis was used to identify differentially regulated genes. Such genes were then ranked by fold change and submitted to the online database, DAVID. The Sprague-Dawley list returned the term "response to hypoxia," absent from the Fischer 344 output. Manual analysis indicated that many genes known to be upregulated by hypoxia-inducible factor-1alpha were downregulated by cyclic hyperoxia. Quantitative real-time RT-PCR analysis of Egln3, Bnip3, Slc16a3, and Hk2 confirmed the microarray results. We conclude that combined methodologies are required for adequate dissection of the pathophysiology of strain susceptibility to OIR in the rat. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s12177-009-9041-7) contains supplementary material, which is available to authorized users.

PCR amplification of the functional immunoglobulin heavy chain variable gene from a hybridoma in the presence of two aberrant transcripts.

Single chain antibody fragment genes are commonly created by splicing together the immunoglobulin light chain (VL) and heavy chain variable (VH) genes of a monoclonal antibody produced by a hybridoma. Selective PCR amplification of the functional immunoglobulin variable gene rearrangements can be complicated by the existence of other unproductive immunoglobulin gene rearrangements in the hybridoma. Here we report the detection and preferential amplification of aberrant transcripts from two unproductive VH gene rearrangements derived from the fusion partner of a hybridoma. The functional VH gene of the monoclonal antibody was successfully amplified by selective use of primers to individual JH segments.

Function and expression of melatonin receptors on human pancreatic islets.

Melatonin is known to inhibit insulin secretion from rodent beta-cells through interactions with cell-surface MT1 and/or MT2 receptors, but the function of this hormone in human islets of Langerhans is not known. In the current study, melatonin receptor expression by human islets was examined by reverse transcription-polymerase chain reaction (RT-PCR) and the effects of exogenous melatonin on intracellular calcium ([Ca2+]i) levels and islet hormone secretion were determined by single cell microfluorimetry and radioimmunoassay, respectively. RT-PCR amplifications indicated that human islets express mRNAs coding for MT1 and MT2 melatonin receptors, although MT2 mRNA expression was very low. Analysis of MT1 receptor mRNA expression at the single cell level indicated that it was expressed by human islet alpha-cells, but not by beta-cells. Exogenous melatonin stimulated increases in intracellular calcium ([Ca2+]i) in dissociated human islet cells, and stimulated glucagon secretion from perifused human islets. It also stimulated insulin secretion and this was most probably a consequence of glucagon acting in a paracrine fashion to stimulate beta-cells as the MT1 receptor was absent in beta-cells. Melatonin did not decrease 3', 5'-cyclic adenosine monophosphate (cyclic AMP) levels in human islets, but it inhibited cyclic AMP in the mouse insulinoma (MIN6) beta-cell line and it also inhibited glucose-stimulated insulin secretion from MIN6 cells. These data suggest that melatonin has direct stimulatory effects at human islet alpha-cells and that it stimulates insulin secretion as a consequence of elevated glucagon release. This study also indicates that the effects of melatonin are species-specific with primarily an inhibitory role in rodent beta-cells and a stimulatory effect in human islets.

Stability of housekeeping gene expression in the rat retina during exposure to cyclic hyperoxia.

Recent evidence suggests a genetic component to oxygen-induced retinopathy (OIR), a robust experimental model of human retinopathy of prematurity. OIR lends itself well to quantitative analysis of gene expression in rodents with well-defined genetic backgrounds. Such analysis by real-time reverse transcription polymerase chain reaction (RT-PCR) requires the use of reference genes as internal standards for purposes of normalization. We sought to identify housekeeping genes showing stable retinal expression across different rat strains and developmental stages, that were not regulated by oxygen tension. Real-time RT-PCR was used to examine in normal (control) neonatal rat retina the expression of five candidate reference genes: acidic ribosomal phosphoprotein (ARBP), cyclophilin A (CYCA), gamma 2 actin (ACTG2), hypoxanthine guanine phosphoribosyltransferase (HPRT), and RNA polymerase 2 (RNAP2). ACTG2 was poorly expressed, whereas quantification of CYCA was confounded by putative amplification of pseudogenes. Expression of ARBP, HPRT, and RNAP2 was then quantified in dissected retinas from neonatal rats of three inbred strains (Fischer 344, Sprague Dawley, and Dark Agouti) under two different conditions of exposure to inspired oxygen (exposure to room air for 14 days from birth; exposure to cyclic hyperoxia for 14 days from birth). The average variation in relative expression between each pair of these three genes within each of the six cDNA test samples was used to assess stability of gene expression, relative to a standard retinal cDNA pool. The relative expression values for ARBP and HPRT were more closely correlated (r2=0.80) than were those for either gene with RNAP2 (ARBP and RNAP2: r2=0.31; HPRT and RNAP2: r2=0.25). There was little variation among the six experimental groups for the normalized expression of ARBP or HPRT (p>0.05). In contrast, the normalized expression of RNAP2 varied significantly amongst experimental groups: Within each strain, expression was higher in the oxygen-exposed group than in the room air-exposed group (p<0.05). We conclude that ARBP and HPRT exhibit expression that is sufficiently stable under conditions of varying oxygen tension, to permit their use as housekeeping genes in at least one model of OIR in the neonatal rat.

Kinetics of strain-dependent differential gene expression in oxygen-induced retinopathy in the rat.

Recent evidence suggests that retinopathy of prematurity, a potentially blinding condition of premature human neonates, has a genetically-determined component. Different inbred strains of rat exhibit differential susceptibility to oxygen-induced retinopathy (OIR), a well-established experimental model of retinopathy of prematurity. To explore the basis for this differential susceptibility, we quantified the retinal expression of 8 angiogenesis-related genes during early post-natal retinal development in rats with OIR. Inbred Fischer 344 (F344), Dark Agouti (DA) and Sprague Dawley (SPD) rat neonates were exposed to alternating cycles of 80% oxygen in air and normoxia for up to 14 days. After 14 days of cyclic hyperoxic exposure, some rats were exposed to normoxia for a further 4 days. Retinal mRNA for vascular endothelial growth factor (VEGF), VEGF receptor 2 (VEGFR2), pigment epithelium-derived factor (PEDF), angiopoietin-2 (Ang2), Tie2, cyclooxygenase-2 (COX2), insulin-like growth factor-1 (IGF1) and erythropoietin (EPO) were quantified by real-time reverse-transcriptase polymerase chain reaction at different time-points. Time-course analysis showed that expression of mRNA for VEGF, VEGFR2 and Ang2 was significantly greater in OIR-resistant (F344) retinae than in OIR-susceptible (DA) retinae during the first 9 days of cyclic hyperoxia. However, at post-natal days 14 and 18, retinal mRNAs for VEGF, EPO, VEGFR2, Ang2, IGF1, COX2 and PEDF were expressed to a significantly greater extent in OIR-susceptible (DA, SPD) than OIR-resistant (F344) retinae. The VEGF/PEDF ratio was greater in the F344 compared with the DA strain up to day 9, but was higher in the DA than the F344 strain at days 14 and 18. Thus, we found that retinal expression of angiogenesis-related genes was significantly higher in OIR-resistant rats than in OIR-susceptible rats during early retinal development, but the pattern reversed during the proliferative phase of OIR. We conclude that susceptibility to OIR correlates with differential gene expression very early in retinal microvascular development, during periods of cyclic hyperoxic exposure rather than during subsequent sustained hypoxia.

Islet alpha-cells do not influence insulin secretion from beta-cells through cell-cell contact.

Interactions between the endocrine cells in islets of Langerhans influence their secretory function, and disruption of islet structure results in impaired insulin secretory responses to both nutrient and non-nutrient stimuli. We have previously demonstrated that insulin-secreting MIN6 cells show enhanced secretory responses when grown as islet-like structures (pseudoislets) suggesting that homotypic cell-cell interactions between beta-cells are important for normal function. We have now extended this experimental model to study the role of heterotypic interactions between insulin-expressing and glucagon-expressing cells by measuring the organization and secretory function of pseudoislets formed from MIN6 and alphaTC1 cells. The direct alpha-cell to beta-cell contact in the heterogenous MIN6/alphaTC1 pseudoislets was sufficient to enable the formation of anatomically correct islet-like structures, with a central core of MIN6 cells surrounded by a periphery of alphaTC1 cells. However, the presence of alphaTC1 cells had no detectable effect on insulin secretory responses to nutrient or non-nutrient stimuli. In contrast, exogenous glucagon enhanced insulin secretion, in accordance with a paracrine role for alpha-cell-derived glucagon in the regulation of insulin secretion rather than direct, contact-mediated effects of alpha-cells on neighbouring beta-cells.

Genetic influences on susceptibility to oxygen-induced retinopathy.

PURPOSE: To investigate the inheritance of susceptibility to oxygen-induced retinopathy in the rat with the use of formal backcross analysis. METHODS: Neonatal offspring of inbred albino Fischer 344 (F344) and pigmented Dark Agouti (DA) crosses and F1xF344 and F1xDA backcrosses were exposed to alternating 24-hour cycles of hyperoxia (80% oxygen in air) and normoxia (21% oxygen in air) for 14 days. Retinal avascular area was analyzed by staining with Griffonia simplicifolia isolectin B4, a marker of vascular endothelial cells. Expression of erythropoietin (EPO) mRNA in retinas was quantified by real-time reverse-transcription polymerase chain reaction. RESULTS: Oxygen-exposed offspring of two F344xDA F1 crosses showed retinal avascular areas and ocular and coat pigmentation that were similar to those of the DA strain. Mean retinal avascular area was 73%. Offspring of two DAxF1 backcrosses were similar to F344xDA F1 pups, with pigmented eyes and coats and a mean retinal avascular area of 76%. In contrast, offspring of two F344xF1 backcrosses exhibited a range of eye and coat pigmentation. Mean retinal avascular area of pigmented offspring of the F344xF1 backcrosses was 71% (P < 0.001 compared with F344 rats). Mean avascular area of albino offspring of the F344xF1 backcrosses was 27% (P > 0.05 compared with F344 rats). The normalized expression of EPO mRNA was 3.01 +/- 1.00 in retinas from pigmented F344xF1 backcross offspring compared with 1.31 +/- 0.69 for albino offspring (P < 0.001). CONCLUSIONS: Segregation of the susceptibility trait to oxygen-induced retinopathy in the DA and F344 rat strains is associated with pigmentation and erythropoietin expression and can be modeled using an autosomal dominant pattern of inheritance.

Single chain antibody fragments for ocular use produced at high levels in a commercial wheat variety.

We are investigating the use of single chain antibody fragments (scFv) in eye drops for diagnosis and treatment of eye diseases. For ocular use, recombinant proteins must be free of bacterial endotoxin that causes inflammation in the eye. We required a means of generating high yields of scFvs with little endotoxin contamination. Using microprojectile bombardment we produced transgenic lines of the commercial wheat variety, Westonia, that express two scFvs that bind to CD4 or CD28 on the surface of rat thymocytes. A high level of expression of active scFv in the range 50-180 microg/g was measured by quantitative flow cytometry in crude extracts made from mature seeds. The levels of expression were stable over four generations of transgenic plants and mature seeds were stored for one year with little loss of scFv activity. Substantial purification of scFv was achieved by immobilised metal affinity chromatography. Compared to bacterial extracts, crude transgenic seed extracts contained only a small amount of endotoxin (150 EU/ml) that will be easily removed by purification. The transgenic wheat lines express functional scFv at levels comparable to production in bacteria and promise to be superior to bacteria for production of scFv pharmaceuticals for ocular use.

Homotypic cell contact enhances insulin but not glucagon secretion.

Intra-islet interactions influence beta-cell function, and disruption of islet architecture results in a reduction in glucose-induced insulin secretion, whereas re-aggregation improves secretory responsiveness. Our studies on MIN6 cells have shown that by configuring beta-cells as three-dimensional islet-like structures there is a marked improvement in glucose-induced insulin secretion compared to that of their monolayer equivalents. In the present study, we have used the mouse glucagon-secreting alphaTC1 cell line to see whether homotypic interactions are important in the regulation of glucagon secretion from alpha-cells. We found no significant difference in the secretory responses of alphaTC1 cells maintained as monolayers or as cell clusters. We also found that different cell adhesion molecules are involved in cell interactions between alpha- and beta-cells; MIN6 cells express ECAD, whereas alphaTC1 cells express NCAM. ECAD is necessary for cell cluster formation by MIN6 cells but not by alphaTC1 cells, whereas NCAM is not needed for the formation of cell clusters in either cell line.

Antigens of selected Acanthamoeba species detected with monoclonal antibodies.

Acanthamoeba species are ubiquitous soil and freshwater protozoa that have been associated with infections of the human brain, skin, lungs and eyes. Our aim was to develop specific antibodies to aid in rapid and specific diagnosis of clinically important isolates. Mice were variously immunised with live mixtures of Acanthamoeba castellanii strain 112 (AC112) trophozoites and cysts, or with sonicated, formalin-fixed or heat-treated trophozoites, or with a trophozoite membrane preparation. Eight hybridoma cell lines secreting monoclonal antibodies reactive with A. castellanii epitopes were generated. Seven of the new antibodies (designated AMEC1-3 and MTAC1-4) were isotyped as IgMkappa and one (MTAC5) as IgG1kappa. All of the novel antibodies bound to AC112 cysts, and MTAC4 and MTAC5 also bound to trophozoites as measured by flow cytometry on unfixed cells. Single chain antibody fragments that retained parental antibody binding characteristics were engineered from three of the hybridomas (AMEC1, MTAC3 and MTAC4). Four monoclonal antibodies (AMEC1, AMEC3, MTAC1, MTAC3) bound reliably to unfixed cysts of clinical isolates of A. castellanii (two strains) and Acanthamoeba polyphaga (two strains), belonging to Pussard-Pons morphological group II, and to Acanthamoeba lenticulata and Acanthamoeba culbertsoni, belonging to Pussard-Pons morphological group III. None of the antibodies bound to cysts or trophozoites of the environmental group I species, Acanthamoeba tubiashi. Antibodies AMEC1, MTAC3, MTAC4 and MTAC5 reacted with buffered formalin-fixed AC112 by immunohistochemistry, and also stained Acanthamoeba in sections of infected rat cornea and buffered formalin-fixed, paraffin-embedded infected human cornea. These antibodies may be useful in diagnosing pathogenic Acanthamoeba species in clinical specimens, provided that cysts are present.