Transcriptome and metabolome profiling in different stages of infestation of Eucalyptus urophylla clones by Ralstonia solanacearum.

Eucalyptus urophylla is an economically important tree species that widely planted in tropical and sub-tropical areas around the world, which suffers significant losses due to Ralstonia solanacearum. However, little is known about the molecular mechanism of pathogen-response of Eucalyptus. We collected the vascular tissues of a E. urophylla clone infected by R. solanacearum in the laboratory, and combined transcriptome and metabolome analysis to investigate the defense responses of Eucalyptus. A total of 11 flavonoids that differentially accumulated at the first stage or a later stage after infection. The phenylpropanoid of p-coumaraldehyde, the two alkaloids trigonelline and DL-ephedrine, two types of traditional Chinese medicine with patchouli alcohol and 3-dihydrocadambine, and the amino acid phenylalanine were differentially accumulated after infection, which could be biomarkers indicating a response to R. solanacearum. Differentially expressed genes involved in plant hormone signal transduction, phenylpropanoids, flavonoids, mitogen-activated protein kinase (MAPK) signaling, and amino acid metabolism were activated at the first stage of infection or a later stage, indicating that they may participate in the defense against infection. This study is expected to deliver several insights into the molecular mechanism in response to pathogens in E. urophylla, and the findings have far-reaching implications in the control of E. urophylla pathogens.

Clone and Function Verification of the OPR gene in Brassica napus Related to Linoleic Acid Synthesis.

BACKGROUND: Fatty acid composition and content affect rapeseed oil quality. Fatty acid synthesis-related genes in rapeseed have been studied globally by researchers. Nevertheless, rapeseed oil is mainly composed of seven different fatty acids (FA), and each fatty acid was regulated by different genes. Furthermore, different FA affect each other, which needs continuous and in-depth research to obtain more clear results in Brassica napus. RESULTS: In this paper, broad-scale miRNA expression profiles were constructed and 21 differentially expressed miRNAs were detected. GO enrichment analysis showed that most up-regulated proteins were involved in transcription factor activity and catalytic activity. KEGG pathway enrichment analysis indicated that 20 pathways involving 36 target genes were enriched, of which the bna00592 pathway may be involved in fatty acid metabolism. The results were verified using a quantitative real-time PCR (RT-qPCR) analysis, we found that the target gene of bna-miR156b > c > g was the OPR (12-oxo-phytodienoic acid reductase). Four copies of OPR gene were found, and the over-expression vectors (pCAMBIA1300-35 s-OPR and pCAMBIA1300-RNAi-OPR) were constructed to verify their functions. In T1 and T2 generation, the content of linoleic acid (LA) increased significantly in OE but deceased in OPRi. CONCLUSIONS: This is the first study to provide four copies of the OPR gene that regulates LA metabolism, can be used for the molecular mechanism of LA and optimizing fatty acid profiles in oilseed for breeding programs.

In vivo clonal analysis reveals spatiotemporal regulation of thalamic nucleogenesis.

The thalamus, a crucial regulator of cortical functions, is composed of many nuclei arranged in a spatially complex pattern. Thalamic neurogenesis occurs over a short period during mammalian embryonic development. These features have hampered the effort to understand how regionalization, cell divisions, and fate specification are coordinated and produce a wide array of nuclei that exhibit distinct patterns of gene expression and functions. Here, we performed in vivo clonal analysis to track the divisions of individual progenitor cells and spatial allocation of their progeny in the developing mouse thalamus. Quantitative analysis of clone compositions revealed evidence for sequential generation of distinct sets of thalamic nuclei based on the location of the founder progenitor cells. Furthermore, we identified intermediate progenitor cells that produced neurons populating more than one thalamic nuclei, indicating a prolonged specification of nuclear fate. Our study reveals an organizational principle that governs the spatial and temporal progression of cell divisions and fate specification and provides a framework for studying cellular heterogeneity and connectivity in the mammalian thalamus.

A strategy to accelerate protein production from a pool of clones in Chinese hamster ovary cells for toxicology studies.

In the biopharmaceutical industry, a clonally derived cell line is typically used to generate material for investigational new drug (IND)-enabling toxicology studies. The same cell line is then used to generate material for clinical studies. If a pool of clones can be used to produce material for IND-enabling toxicology studies (Pool for Tox (PFT) strategy) during the time a lead clone is being selected for clinical material production, the toxicology studies can be accelerated significantly (approximately 4 months at Genentech), leading to a potential acceleration of 4 months for the IND submission. We explored the feasibility of the PFT strategy with three antibodies-mAb1, mAb2, and mAb3-at the 2 L scale. For each antibody, two lead cell lines were identified that generated material with similar product quality to the material generated from the associated pool. For two antibody molecules, mAb1 and mAb2, the material generated by the lead cell lines from 2 L bioreactors was tested in an accelerated stability study and was shown to have stability comparable to the material generated by the associated pool. Additionally, we used this approach for two antibody molecules, mAb4 and mAb5, at Tox and GMP production. The materials from the Tox batch at 400 L scale and three GMP batches at 2000 L scale have comparable product quality attributes for both molecules. Our results demonstrate the feasibility of using a pool of clonally derived cell lines to generate material of similar product quality and stability for use in IND-enabling toxicology studies as was derived from the final production clone, which enabled significant acceleration of timelines into clinical development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1449-1455, 2017.

Microdissection and visualization of individual hair follicles for lineage tracing studies.

In vivo lineage tracing is a valuable technique to study cellular behavior. Our lab developed a lineage tracing method, based on the Cre/lox system, to genetically induce clonal labelling of cells and follow their progeny. Here we describe a protocol for temporally controlled clonal labelling and for microdissection of individual mouse hair follicles. We further present staining and visualization techniques used in our lab to analyze clones issued from genetically induced labelling.

Deletion and anergy of polyclonal B cells specific for ubiquitous membrane-bound self-antigen.

B cell tolerance to self-antigen is critical to preventing antibody-mediated autoimmunity. Previous work using B cell antigen receptor transgenic animals suggested that self-antigen-specific B cells are either deleted from the repertoire, enter a state of diminished function termed anergy, or are ignorant to the presence of self-antigen. These mechanisms have not been assessed in a normal polyclonal repertoire because of an inability to detect rare antigen-specific B cells. Using a novel detection and enrichment strategy to assess polyclonal self-antigen-specific B cells, we find no evidence of deletion or anergy of cells specific for antigen not bound to membrane, and tolerance to these types of antigens appears to be largely maintained by the absence of T cell help. In contrast, a combination of deleting cells expressing receptors with high affinity for antigen with anergy of the undeleted lower affinity cells maintains tolerance to ubiquitous membrane-bound self-antigens.

Screening for novel lead compounds increasing insulin expression in medullary thymic epithelial cells.

Insulin expression in the thymus has been implicated in regulating the negative selection of autoreactive T cells and in mediating the central immune tolerance to pancreatic beta-cells. Thymic insulin expression modulation might be an important drug target for preventing type 1 diabetes. We performed a high-throughput screening to identify compounds with such activity. A reporter plasmid was constructed with the human insulin promoter sequence including a short allele of the upstream variable number tandem repeat (VNTR) sequence (32 repeats), subcloned into the pGL4.17 vector. The plasmid was stably transfected into an insulin-transcribing medullary thymic epithelial cell (mTEC) line. Primary high-throughput screening assays were carried out by stimulating with candidate compounds for 24h, and the activity of luciferase was measured. Positive compounds were further validated by real-time PCR. Of 19,707 compounds, we identified one compound that could enhance mTEC insulin expression, as confirmed by real-time PCR. We also observed that transfection with the autoimmune regulator (AIRE) increased endogenous AIRE expression in mTECs. Our insulin-VNTRI-promoter reporter system is consistent with the insulin expression regulation in mTECs, and one compound that was identified could increase insulin expression in mTECs. A positive feedback effect of AIRE in mTECS was observed. Whether these efforts in murine thymus cells apply to humans remains to be determined.

Induction of antioxidant and phase 2 drug-metabolizing enzymes by falcarindiol isolated from Notopterygium incisum extract, which activates the Nrf2/ARE pathway, leads to cytoprotection against oxidative and electrophilic stress.

In the present study, we isolated falcarindiol from Notopterygium incisum and investigated the effect of falcarindiol on the expression of antioxidant enzymes (AOEs), such as catalase, and phase 2 drug-metabolizing enzymes (DMEs), such as glutathione S-transferase and NAD(P)H:quinone oxidoreductase 1, in a cultured cell line from normal rat liver, Clone 9 cells. Exposure of Clone 9 cells to falcarindiol resulted in the significant induction of AOEs and phase 2 DMEs. Western blot analysis and transfection studies using a luciferase reporter construct demonstrated that the induction of AOEs and phase 2 DMEs by falcarindiol was caused through the Nrf2/ARE (nuclear factor-E2-related factor 2/antioxidant response element) pathway. Pretreatment of cells with falcarindiol accelerated the detoxification of a potentially toxic quinone (menadione) and mitigated menadione-induced cytotoxicity. We found that falcarindiol was a novel inducer of AOEs and phase 2 DMEs and falcarindiol might exhibit chemopreventive activity.

Recognition of T cell epitopes unique to Cha o 2, the major allergen in Japanese cypress pollen, in allergic patients cross-reactive to Japanese cedar and Japanese cypress pollen.

BACKGROUND: Pollens from species of the Cupressaceae family are one of the most important causes of respiratory allergies worldwide. Many patients with pollinosis have specific IgE to both allergens from Japanese cedar and Japanese cypress pollen. We set out to identify T cell epitopes in Cha o 2, the second major allergen of Japanese cypress pollen. METHODS: T cell lines (TCL) and T cell clones (TCC) specific to Cha o 2 were generated from allergic patients cross-reactive to Japanese cedar and Japanese cypress pollen. T cell epitopes in Cha o 2 were identified by responses of TCL stimulated with overlapping peptides. Abilities of IL-4/IFN-gamma production by TCC were evaluated using enzyme immunoassay. RESULTS: Using TCL, 11 dominant and subdominant T cell epitopes were identified in Cha o 2. The subsets of TCC were predominantly of T helper 2-type. A T cell epitope p141-160 in Cha o 2 and corresponding peptide in Cry j 2 showed high homology. Although TCC PC.205.159 responded to stimulation with p141-160 in Cha o 2, it did not respond with corresponding peptide in Cry j 2, therefore, the T cell epitope was unique to Cha o 2. CONCLUSIONS: Eleven T cell epitopes that were identified are unique to Cha o 2. Cha o 2 is a putative aeroallergen that can potentially sensitize human T cells. We concluded that generation of T cells specific to Cha o 2 in allergic patients acts as one of the causes of continuous allergic symptoms in April.

Cysteine-string protein isoform beta (Cspbeta) is targeted to the trans-Golgi network as a non-palmitoylated CSP in clonal beta-cells.

Cysteine string proteins (CSPs) belong to the DnaJ-like chaperone family and play an important role in regulated exocytosis in neurons and endocrine cells. The palmitoylation of several residues in a cysteine string domain may anchor CSPs to the exocytotic vesicle surface and in pancreatic beta-cells, Cspalpha is localized on insulin containing large dense core vesicles (LDCVs). An isoform closely related to Cspalpha, Cspbeta, has been obtained from testis cell cDNA libraries. To gain insights on this isoform and more generally on the properties of CSPs, we compared Cspalpha and Cspbeta. In pull-down experiments, Cspbeta was able to interact to the same extent with two of the known Cspalpha chaperone partners, Hsc70 and SGT. Upon transient overexpression in clonal beta-cells, Cspbeta but not Cspalpha was mainly produced as a non-palmitoylated protein and mutational analysis indicated that domains distinct from the cysteine string are responsible for this difference. As Cspbeta remained tightly bound to membranes, intrinsic properties of CSPs are sufficient for interactions with membranes. Indeed, recombinant Cspalpha and Cspbeta were capable to interact with membranes even in their non-palmitoylated forms. Furthermore, overexpressed Cspbeta was not associated with LDCVs, but was localized at the trans-Golgi network. Our results suggest a possible correlation between the specific membrane targeting and the palmitoylation level of CSPs.

Significant variations in differentiation properties between independent mouse ES cell lines cultured under defined conditions.

Mouse embryonic stem (ES) cells are isolated from the inner cell mass (ICM)/epiblast of preimplantation embryos and are widely used in cell differentiation studies. We have previously observed differences in transcript and antigen expression following differentiation of ES cells lines in vitro. We have investigated this further by comparing the differentiation characteristics of five independently derived ES cell lines cultured and differentiated under defined conditions. Undifferentiated ES cell lines exhibited similar morphology and antigen/transcript marker expression. However, upon differentiation in monolayer culture by LIF withdrawal, only two of the lines expressed similar germ layer transcript profiles, and these were significantly altered compared to differentiation in serum-supplemented media. Neurofilament-68k was the only transcript marker common to all cell lines, however, induction of neuroectoderm lineages using 1 microM all-trans retinoic acid (RA) resulted in significant variations in cell number and morphology between the lines. Furthermore, neurons were only formed from clones of the two cell lines that exhibited similar transcript profiles, although the morphology was different between the two. We conclude that the independent ES cell lines in this study differ in their response to alterations in culture conditions in vitro, and the use of an appropriate cell line enables relatively homogeneous neuronal populations to be achieved in monolayer culture under defined conditions.

Decreased susceptibility of leukemic cells with PIG-A mutation to natural killer cells in vitro.

The cloning of the PIG-A gene has facilitated the unraveling of the complex pathophysiology of paroxysmal nocturnal hemoglobinuria (PNH). Of current major concern is the mechanism by which a PNH clone expands. Many reports have suggested that an immune mechanism operates to cause bone marrow failure in some patients with PNH, aplastic anemia, and myelodysplastic syndromes. Because blood cells of PNH phenotype are often found in patients with these marrow diseases, one hypothesis is that the PNH clone escapes immune attack, producing a survival advantage by immunoselection. To test this hypothesis, we examined the sensitivity of blood cells, with or without PIG-A mutations, to killing by natural killer (NK) cells, using 51Cr-release assay in vitro. To both peripheral blood and cultured NK cells, PIG-A mutant cells prepared from myeloid and lymphoid leukemic cell lines were less susceptible than their control counterparts (reverted from the mutant cells by transfection with a PIG-A cDNA). NK activity was completely abolished with concanamycin A and by calcium chelation, indicating that killing was perforin-dependent. There were no differences in major histocompatibility (MHC) class I expression or sensitivity to either purified perforin or to interleukin-2-activated NK cells between PIG-A mutant and control cells. From these results, we infer that PIG-A mutant cells lack molecules needed for NK activation or to trigger perforin-mediated killing. Our experiments suggest that PIG-A mutations confer a relative survival advantage to a PNH clone, contributing to selective expansion of these cells in the setting of marrow injury by cytotoxic lymphocytes.

Gamma-glutamyl transferase expression in higher-grade astrocytic glioma.

Increased expression of gamma-glutamyltransferase (GGT) has been detected in a range of human malignancies and is thought to be involved in neoplastic proliferation and treatment resistance. Since GGT expression and its role in malignant glioma biology remain largely unknown, we investigated this phenomenon by immunostaining 26 higher-grade human astrocytic gliomas (WHO grades III and IV) with a monoclonal anti-GGT-antibody (138H11). Further, human pancreatic GGT cDNA was used for liposome-mediated transfection of 9L gliosarcoma cells. GGT-expressing and control 9L cells were cultured in media containing different amounts of essential amino acids and/or cytotoxic agents. Cell viability was evaluated by microplate MTT assay. Immunohistochemical staining of tumor specimens demonstrated that GGT expression is a frequent feature of higher-grade human astrocytic gliomas, but not of normal brain tissue. Human tumors were strongly GGT-positive in 6 of 7 cases of grade III astrocytoma, and in 12 of 19 grade IV astrocytoma (glioblastoma multiforme, GBM) cases. In the cell culture model, 9L-GGT cells had a growth advantage over control cells in cysteine-deficient medium. but not in standard or glutamine-free medium. No significant difference in numbers of viable cells of either clone was found in media containing the alkylating drug BCNU (5-200 microg/ml). In conclusion, GGT is expressed in a high percentage of human WHO grade III astrocytomas and GBM, but not in normal brain tissue. This molecule seems to give neoplastic cells a moderate growth advantage under in vivo conditions.

Migration and multipotentiality of PSA-NCAM+ neural precursors transplanted in the developing brain.

By optimizing the previously described strategy for obtention of spheres enriched in PSA-NCAM+ precursors, we prepared PSA-NCAM-immunoselected cell populations from cerebral hemispheres of neonatal MBP-LacZ transgenic mice. These cells expressed Nestin, exhibited clonal expansion potential and formed spheres, which were initially enriched in PSA-NCAM+ cells but became enriched in GD3+ oligodendrocyte progenitors after 1 week in B104 contionned medium. One month after their periventricular transplantation into the brain of wild-type and/or shiverer newborn mice, cells from PSA-NCAM+ spheres exhibited a higher rostral migration potential than cells from GD3+ spheres, and clearly contributed to myelination in the olfactory bulb. In shiverer hosts, both sphere populations generated oligodendrocytes with similar myelination potential. In addition PSA-NCAM+ sphere cells generated GFAP+ astrocytes and NeuN+ neurons, depending on their site of insertion. These results evidence the high plasticity of newborn PSA-NCAM+ neural precursors and suggest that they are promising tools for cell therapy of CNS diseases, including myelin disorders.

Inhibitors of human cytomegalovirus replication drastically reduce the activity of the viral protein kinase pUL97.

The UL97-encoded protein kinase (pUL97) of human cytomegalovirus (HCMV) plays a critical role in the control of virus replication. Deletion of the UL97 gene results in a drastic reduction in the replication efficiency. Although the exact function of pUL97 remains unclear and its sensitivity to specific inhibitors is speculative, protein kinase inhibitors of the indolocarbazole class are effective inhibitors of cytomegalovirus. Based on the phosphorylation of ganciclovir (GCV), a novel quantification system for pUL97 kinase activity was established: the phosphorylated form of GCV exerts an easily quantifiable cytotoxic effect in transfected cells. Importantly, the addition of indolocarbazole compounds, Gö6976 and NGIC-I, which were highly effective at nanomolar concentrations while other protein kinase inhibitors were not, led to a significant reduction of pUL97 kinase activity. It was also demonstrated that a catalytically inactive mutant of pUL97, K355M, and a GCV-resistant mutant, M460I, were both negative for GCV phosphorylation, although protein phosphorylation remained detectable for the latter mutant. In vitro kinase assays were used to confirm the levels of pUL97-mediated phosphorylation recorded. To generate a tool for screening large numbers of putative inhibitors that preferentially interfere with GCV as well as protein phosphorylation, pUL97-expressing cell clones with stable pUL97 kinase activity were selected. This study demonstrates that certain indolocarbazole compounds are potent pUL97 inhibitors and, therefore, represent novel candidates for antiviral drugs that target viral protein kinase functions.

Identification of Th2-type suppressor T cells among in vivo expanded ocular T cells in mice with experimental autoimmune uveoretinitis.

Experimental autoimmune uveoretinitis (EAU), which is a T cell mediated organ specific autoimmune disease, is induced by immunization with interphotoreceptor retinoid binding protein (IRBP) in susceptible strains of mice. It has been found that IRBP-derived peptide 518-529 (p518-529) generates Th2-type responses and inhibits IRBP-induced EAU, indicating that the p518-529 might be an epitope for suppressor T cells in IRBP-induced EAU. First, we observed that there were T cells producing the Th2 type cytokines such as IL-4 and IL-10 in late phase of EAU. Furthermore, to examine whether p518-529-reactive T cells expand in the eye during EAU, T cell receptor (TCR) of ocular T cells was compared with that of p518-529 reactive T cells in spleen from mice with EAU by PCR-single strand conformation polymorphism (PCR-SSCP) and nucleotide sequence analysis. SSCP and sequence analyses indicated that p518-529 reactive TCR BV10+ T cells bearing amino acid motif(PWG) and TCR BV13+ T cells bearing amino acid motif(PGLGGY) in their complementary-determining region 3 (CDR3) region were clonally expanding in ocular tissues on day 28 after immunization, although these T cells were not detected on day 14. These findings demonstrate that p518-529 reactive Th2-type T cells expand oligoclonally in the uveitic eyes in the late stage of EAU and may function as Th2-type suppressor T cells for improvement of the disease.

Antisense inhibition of BCL-2 expression induces retinoic acid-mediated cell death during differentiation of human NT2N neurons.

Changes in expression of the proto-oncogene Bcl-2 are well known in the developing brain, with a high expression level in young post-mitotic neurons that are beginning the outgrowth of processes. The physiological significance of the Bcl-2 up-regulation in these neurons is not fully understood. We used a differentiation model for human CNS neurons to study the expression and function of Bcl-2. NT2/D1 human neuronal precursor cells differentiated into a neuronal phenotype in the presence of 10 microM retinoic acid for 3-5 weeks. This concentration of retinoic acid was not toxic to undifferentiated NT2/D1 cells but was sufficient to up-regulate the BCL-2 protein in 6 days. The BCL-2 levels increased further after 3 weeks, i.e. when the cells started to show neuronal morphology. Inhibition of the accumulation of endogenous BCL-2 with vectors expressing the antisense mRNA of Bcl-2 caused extensive apoptosis after 3 weeks of the retinoic acid treatment. The loss of neuron-like cells from differentiating cultures indicated that the dead cells were those committed to neuronal differentiation. Death was related to the presence of retinoic acid since withdrawal of retinoic acid after 16 days of treatment dramatically increased cell surviving. The ability of BCL-2 to prevent retinoic acid-induced cell death was also confirmed in undifferentiated NT2/D1 cells that were transfected with a vector containing Bcl-2 cDNA in sense orientation and exposed to toxic doses (40-80 microM) of retinoic acid. Furthermore, down-regulation of BCL-2 levels by an antisense oligonucleotide in neuronally differentiated NT2/D1 cells increased their susceptibility to retinoic acid-induced apoptosis. These results indicate that one function of the up-regulation of endogenous BCL-2 during neuronal differentiation is to regulate the sensitivity of young post-mitotic neurons to retinoic acid-mediated apoptosis.

Crucial role of type 1, but not type 3, inositol 1,4,5-trisphosphate (IP(3)) receptors in IP(3)-induced Ca(2+) release, capacitative Ca(2+) entry, and proliferation of A7r5 vascular smooth muscle cells.

Stimulation of G protein- or tyrosine kinase-coupled receptors regulates cell proliferation through intracellular Ca(2+) ([Ca(2+)](i)) signaling. In A7r5 cells, we confirmed that inositol 1,4,5-trisphosphate (IP(3)) mediates vasopressin (VP)-evoked Ca(2+) release from intracellular stores and showed that types 1 (IP(3)R(1)) and 3 (IP(3)R(3)) IP(3) receptors were expressed. Using antisera selective for IP(3)R(1) or IP(3)R(3) and another that interacted equally well with both subtypes, together with membranes from SF:9 cells expressing only single IP(3)R subtypes to calibrate immunoblotting, we established that A7r5 cells express 81% IP(3)R(1) and 19% IP(3)R(3). To elucidate the contributions of IP(3)R(1) and IP(3)R(3) to Ca(2+) signaling and proliferation, stable clones expressing promoter-inducible antisense cDNA fragments (-90 to +9) corresponding to the two IP(3)R subtypes were selected. Mild inhibition of IP(3)R(1) (71+/-8% of control level) slightly attenuated the IP(3)-evoked Ca(2+) release (IICR) induced by VP but significantly decreased the subsequent capacitative Ca(2+) entry (CCE) and proliferation. Moderate inhibition (34+/-6%) strongly decreased both IICR and CCE and further blocked proliferation. Complete inhibition almost abolished IICR and CCE and arrested proliferation entirely. Complete inhibition of IP(3)R(3) expression slightly attenuated IICR without affecting CCE or proliferation. In cells microinjected with a low dose of heparin, VP-induced CCE was more susceptible than IICR to mild inhibition of both IP(3)R(1) and IP(3)R(3). A high dose of heparin had a similar effect to complete inhibition of IP(3)R(1) expression: it blocked VP-evoked IICR entirely and CCE by 90%. We conclude that IP(3)R(1), but not IP(3)R(3), is crucial for IICR, CCE, and proliferation of vascular smooth muscle cells.

Development and characterization of pituitary GH3 cell clones stably transfected with a human proinsulin cDNA.

Successful beta-cell replacement therapy in insulin-dependent (type I) diabetes is hindered by the scarcity of human donor tissue and by the recurrence of autoimmune destruction of transplanted beta cells. Availability of non-beta cells, capable of releasing insulin and escaping autoimmune recognition, would therefore be important for diabetes cell therapy. We developed rat pituitary GH3 cells stably transfected with a furin-cleavable human proinsulin cDNA linked to the rat PRL promoter. Two clones (InsGH3/clone 1 and 7) were characterized in vitro with regard to basal and stimulated insulin release and proinsulin transgene expression. Mature insulin secretion was obtained in both clones, accounting for about 40% of total released (pro)insulin-like products. Immunocytochemistry of InsGH3 cells showed a cytoplasmic granular insulin staining that colocalized with secretogranin II (SGII) immunoreactivity. InsGH3 cells/clone 7 contained and released in vitro significantly more insulin than clone 1. Secretagogue-stimulated insulin secretion was observed in both InsGH3 clones either under static or dynamic conditions, indicating that insulin was targeted also to the regulated secretory pathway. Proinsulin mRNA levels were elevated in InsGH3 cells, being significantly higher than in betaTC3 cells. Moreover, proinsulin gene expression increased in response to various stimuli, thereby showing the regulation of the transfected gene at the transcriptional level. In conclusion, these data point to InsGH3 cells as a potential beta-cell surrogate even though additional engineering is required to instruct them to release insulin in response to physiologic stimulations.

Differential regulation of antigen-induced IL-4 and IL-13 generation from T lymphocytes by IFN-alpha.

BACKGROUND: IL-4 and IL-13 are related cytokines with similar functional properties. Differential regulation of IL-4 and IL-13 has not been described. OBJECTIVE: We have examined the effects of IFN-alpha on antigen-driven proliferation, IL-4 generation, and IL-13 generation from human PBMCs and T-cell clones. METHODS: Proliferation was assessed by 3H-thymidine incorporation. Cytokine generation was assessed by reverse transcription PCR and ELISA. Messenger RNA stability was assessed in the presence of actinomycin D. RESULTS: IFN-alpha induced a concentration-dependent inhibition of antigen-driven proliferation of TH1 and TH2 clones (median effective concentration, 150 to 200 U/mL); the sensitivity of TH1 and TH2 clones to IFN-alpha was not significantly different (P =.6). IFN-alpha induced an analogous concentration-dependent inhibition of antigen-driven IL-13 generation from TH1 and TH2 clones (median effective concentration, 100 U/mL); this effect was evident by 12 hours of culture and persisted beyond 48 hours. However, IL-4 generation from TH2 clones was insensitive to IFN-alpha at all concentrations and times tested (1 to 10,000 U/mL). A similar inhibitory effect of IFN-alpha on mitogen-driven proliferation and IL-13 generation from PBMCs was demonstrated; once again, IL-4 generation from PBMCs was insensitive to IFN-alpha. IL-13 mRNA stability was unaffected by IFN-alpha, suggesting transcriptional regulation. CONCLUSION: IFN-alpha differentially regulates antigen-stimulated IL-4 and IL-13 generation.