Modification Of Culture Conditions Research Articles

Overexpression of ovine leptin in Pichia pastoris: physiological yeast response to leptin production and characterization of the recombinant hormone.

Ovine leptin was cloned in the methylotrophic yeast Pichia pastoris using a pPIC9K vector. Leptin was produced and secreted into the culture medium using the Saccharomyces cerevisiae alpha-mating factor prepro signal by five clones. Expression levels of leptin varied from clone to clone, depending on the copy number of the ob gene. Highest expression was observed with the single-copy clone S27 (250 mg/l). The modifications of culture conditions in batch and fed-batch culture increase the yield of protein. The use of higher cell concentration (63 g/l) before induction of oLept associate with a regulation of pH at 3.2, which decreases the effects of proteolysis, increases the expression level of the oLept to 402 mg/l. Moreover, compared with the non-producer clone, we observed a drastic decrease in growth rate and biomass yield in the leptin-producing clones. At the end of the fed-batch phase at pH 3.2 with clone S27, mortality rate reached 17.3%. Results showed that recombinant leptin production induced metabolic stress, and a negative impact on biomass yield and growth rate. We characterized the recombinant leptin produced by clone S27. It exhibited a molecular mass of 16 kDa, an N-terminal amino acid sequence identical to that of ovine leptin but with an additional tyrosine introduced by the cloning site. Moreover, it was found to be biologically active in vitro. The available production of a large quantity of oLept will strengthen the functional study for theoretical and practical purposes.

Agrobacterium rhizogenes-mediated transformation: root cultures as a source of alkaloids.

Hairy roots, transformed with Agrobacterium rhizogenes, have been found to be suitable for the production of secondary metabolites because of their stable and high productivity in hormone-free culture conditions. A number of plant species including many medicinal plants have been successfully transformed with Agrobacterium rhizogenes. Transformed root cultures have also been found to be a potential source of high-value pharmaceuticals. In this article the most important alkaloids produced by hairy roots are summarised. Several different methods have been used to increase the alkaloid accumulation in hairy root cultures. The selection of high productive root lines based on somaclonal variation offers an interesting option to enhance the productivity. Elicitors and modification of culture conditions have been shown to increase the growth and the alkaloid production in some cases. Genetic engineering is a modern tool to regulate the secondary metabolism also in hairy roots. However, our knowledge on biosynthesis of many alkaloids is still poor. Only a limited number of enzymes and their respective genes which regulate the biosynthetic pathways are fully characterised.

Routine detection of Epstein–Barr virus specific T-cells in the peripheral blood by flow cytometry

The ability to detect cytomegalovirus-specific T-cells (CD4 +) in the peripheral blood by flow cytometry has been recently described by Picker et al. In this method, cells are incubated with viral antigen and responding (cytokine producing) T-cells are then identified by flow cytometry. To date, this technique has not been reliably used to detect Epstein–Barr virus (EBV)-specific T-cells primarily due to the superantigen/mitogenic properties of the virus which non-specifically activate T-cells. By modifying culture conditions under which the antigens are presented, we have overcome this limitation and developed an assay to detect and quantitate EBV-specific T-cells. The detection of cytokine producing T-cells by flow cytometry requires an extremely strong signal (such as culture in the presence of PMA and ionomycin). Our data indicate that in modified culture conditions (early removal of viral antigen) the non-specific activation of T-cells by EBV is reduced, but antigen presentation will continue uninhibited. Using this method, EBV-specific T-cells may be legitimately detected using flow cytometry. No reduction in the numbers of antigen-specific T-cells was observed by the early removal of target antigen when verified using cytomegalovirus antigen (a virus with no non-specific T-cell activation properties). In EBV-seropositive individuals, the phenotype of the EBV-specific cytokine producing T-cells was evaluated using four-color flow cytometry and found to be CD45 +, CD3 +, CD4 +, CD45RA −, CD69 +, CD25 −. This phenotype indicates the stimulation of circulating previously unactivated memory T-cells. No cytokine production was observed in CD4 + T-cells from EBV-seronegative individuals, confirming the specificity of this assay. In addition, the use of four color cytometry (CD45, CD3, CD69, IFNγ/IL-2) allows the total quantitative assessment of EBV-specific T-cells while monitoring the interference of EBV non-specific mitogenic activity. This method may have significant utility for the monitoring of the immune response to latent virus infection/reactivation.

Metabolic engineering of indene bioconversion in Rhodococcus sp.

We have applied the methodology of metabolic engineering in the investigation of the enzymatic bioreaction network in Rhodococcus sp. that catalyzes the bioconversion of indene to (2R)-indandiol suitable for the synthesis of cis-1-amino-2-indanol, a precursor of the HIV protease inhibitor, Crixivan. A chemostat with a novel indene air delivery system was developed to facilitate the study of steady state physiology of Rhodococcus sp. 124. Prolonged cultivation of this organism in a continuous flow system led to the evolution of a mutant strain, designated KY1, with improved bioconversion properties, in particular a twofold increase in yield of (2R)-indandiol relative to 124. Induction studies with both strains indicated that KY1 lacked a toluene-inducible dioxygenase activity present in 124 and responsible for the formation of undesired byproducts. Flux analysis of indene bioconversion in KY1 performed using steady state metabolite balancing and labeling with [14C]-tracers revealed that at least 94% of the indene is oxidized by a monooxygenase to indan oxide that is subsequently hydrolyzed to trans-(1R,2R)-indandiol and cis-(1S,2R)-indandiol. This analysis identified several targets in KY1 for increasing (2R)-indandiol product yield. Most promising among them is the selective hydrolysis of indan oxide to trans-(1R,2R)-indandiol through expression of an epoxide hydrolase or modification of culture conditions.

Effect of Photoperiod and Nitrogen Supply on Basal Shoots Development in Rhododendron Catawbiense

In order to control young plant form by modifying culture conditions, plants of Rhododendron catawbiense from in vitro culture were grown in a greenhouse under different photoperiodic treatments (long or short days) combined or not with a several-week nitrogen starvation. After 12 weeks of culture under long days (16 h) with nitrogen supply, plants showed a rhythmic acrotonous development. When long days were combined with a six-week nitrogen starvation, the apical growth pause was extended leading to an increase of the number of acrotonous lateral ramifications. Short-day (8 h) treatment affected distal burst potential and moreover when a concomitant nitrogen starvation was applied. This lack of distal development allowed basal buds swelling, leading to basitonous plants. When plants were returned back to long days after 2, 4 or 6 weeks under short days, distal buds resumption competed with basal shoots development. Durable basitonous plants were obtained by a 12-week short days treatment combined with a 6-week nitrogen starvation.

Modified Culture Conditions Enhance Expression of Differentiated Phenotypic Properties of Normal Rat Cholangiocytes

Modified Culture Conditions Enhance Expression of Differentiated Phenotypic Properties of Normal Rat Cholangiocytes

Dynamic model of ex vivo granulocytic kinetics to examine the effects of oxygen tension, pH, and interleukin-3.

Evaluating kinetics in hematopoietic cultures is complicated by the distribution of cells over various stages of differentiation and by the presence of cells from different lineages. Thus, an observed response is an integral response from distributed cell populations. Growth factors and other parameters can greatly affect the lineage and maturation stage of the culture outcome. To resolve the kinetics and more clearly define the differential effects of O(2) tension (pO(2)), pH, and interleukin-3 (IL-3) on granulopoiesis, a mathematical model-based approach was undertaken. Granulocytic differentiation is described within a continuous, deterministic framework in which cells develop from primitive granulocytic progenitors to mature neutrophils. The model predicts two distributed populations-quiescent and cycling cells-by incorporating rates of growth, death, differentiation, and transition between quiescence and active cycling. The response of these four model processes to changes in the culture environment was examined. Model simulations of experimental data revealed the following: 1) pO(2) effects are exerted only on the growth rate but not maturation times. 2) pH effects between pH 7.25 and 7.4 on growth and differentiation are coupled; however, with increasing pH values, especially at pH 7. 6, the death rate for cells in the early stages of differentiation becomes increasingly significant. 3) The absence of IL-3 increases the death rate for primitive cells only minimally but markedly enhances the rate of differentiation through the myeloblast window in the differentiation pathway. The combined effects of these environmental factors can be predicted based on changes in the model parameters derived from the individual effects. Experimental data combined with mathematical modeling can elucidate the mechanisms underlying the regulation of granulopoiesis by pO(2), pH, and IL-3. The model also can be readily adapted to evaluate the effects of other culture conditions. The increased understanding of experimental results gained with this approach can be used to modify culture conditions to optimize ex vivo production of neutrophil precursors.

Effective ex vivo generation of granulopoietic postprogenitor cells from mobilized peripheral blood CD34 + cells

Objective Neutropenia following high-dose chemotherapy and peripheral blood progenitor cell (PBPC) transplantation might be abrogated by an additional transplantation of ex vivo generated granulopoietic postprogenitor cells (GPPC). Therefore, the ex vivo expansion of CD34 + PBPC was systematically studied aiming for optimum GPPC production. Materials and Methods CD34 + PBPC were cultured in serum-free medium comparing different (n = 32) combinations of stem cell factor (S), interleukin 1 (1), interleukin 3 (IL-3) (3), interleukin-6 (6), erythropoietin (E), granulocyte colony-stimulating factor (G), granulate-macrophage colony-stimulating factor (GM), daniplestim (D, a novel IL-3 receptor agonist), and Flt3 ligand (FL) under various culture conditions. Ex vivo generated cells were assessed by flow cytometry, morphology, and progenitor cell assays. Results Addition of G ± GM but not GM alone to cultures stimulated with S163E effectively induced the generation of GPPC. GPPC production was maximum after 12 to 14 days. Best expansion rates were observed when cells were cultured at 1.5 × 10 4 /mL in 21% O 2. Modifications of culture conditions were either less or equally effective (i.e., modification of starting cell concentrations, low oxygen, addition of serum albumin or autologous plasma, repetitive feeding). Comparison of different cytokine combinations revealed that the optimum GPPC expansion cocktail consisted of S6GD+FL (day 12: 130-fold cellular expansion, 32% myeloblasts /promyelocytes, 49.4% myelocytes /metamyelocytes, 12.4% bands /segmented), which furthermore expanded CD34 + cells (3.4-fold) and clonogenic progenitors (13.4-fold). Conclusion Using the S6DG+FL expansion cocktail, GPPC could be effectively produced ex vivo starting from positively selected CD34 PBPC, possibly enabling amelioration or even abrogation of posttransplant neutropenia.

Biological factors in culture media affecting in vitro fertilization, preimplantation embryo development, and implantation.

Optimal culture conditions are of paramount importance for in vitro fertilization of gametes, preimplantation embryo development, and implantation for all species. Water is the basis of all culture media, and ultrapure water should be employed. The main energy sources of a medium are lactate, pyruvate, and glucose. The concentrations of the first two vary in different media, whereas the latter is necessary mainly for the later stages (morula to blastocyst) of development. A fixed nitrogen source is essential for implantation embryo development whether this is provided by amino acids, albumin, or serum. Suboptimal culture conditions can block development. Pronuclear zygotes of most species (but not human) arrest at some point between the two-cell and the 16-cell stage. Modifying culture conditions can lead the embryos to develop through this block. Hypoxanthine also causes a two-cell block to mouse pronuclear zygotes, and this again depends largely on culture conditions. Simple culture media are bicarbonate-buffered systems with pyruvate, lactate, and glucose. Complex media, such as Ham's F-10, contain in addition amino acids and other elements found in serum. Human tubal fluid simulates the fallopian tube microenvironment. EDTA, gonadotropins, growth factors, and other substances can be included in the media to stimulate development. Coculture of embryos with oviductal cells has shown promising results.

Analysis of light/dark synchronization of cell-wall-less Chlamydomonas reinhardtii (Chlorophyta) cells by flow cytometry

The degree of synchrony of light/dark-adapted Chlamydomonas reinhardtii cell-wall-less cells was analysed by flow cytometry. The cultures exhibited the classical growth curve reported for synchronously dividing cells. However, analysis of the nuclear DNA content revealed a partial asynchrony of cell division. This allowed us to determine that cell division lasts only 1 h 15 min. The changes in the distribution of nuclei in G1 and S phases did not exhibit the DNA peaks expected for highly synchronous cells. However, these peaks were observed for the G2+M nuclei and reflected the number of cell divisions. Modification of culture conditions including temperature, photoperiod, quantum irradiance and culture density affected the degree of synchrony but did not completely abolish the asynchrony. An overlap between the divisions of cells dividing one, two or three times could account for the partial asynchrony observed. In fact, it is possible that cells perform their divisions synchronously but that the timing and/or the duration of the cell divisions is different for the three kinds of cells. The synchrony seems to be perturbed when the cells move from the G1 to the S phase. The transduction of the release signal, allowing cells to pass through the restriction point of the G1 phase, might be too slow and insufficiently homogeneous between the cells to allow perfect synchronization. Finally, if highly synchronized cells are needed to study Chlamydomonas reinhardtii cell cycle, other synchronization procedures should be considered.

A two-dimensional protein map of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are used extensively for the expression of biopharmaceutical protein products. As part of our effort to better understand CHO cell physiology and protein expression changes caused by modified culture conditions, we have begun to map CHO cell polypeptides. A parental cell line reference map was established using two-dimensional gel electrophoresis with immobilized pH gradients (pH 3-10) in the first dimension and a linear acrylamide gradient (9-18%T) in the second dimension. The map is composed of over 1000 silver-stained protein spots. Protein identification is proceeding using a combination of immunostaining, NH2-terminal sequencing, and mass spectrometric analyses. Among the proteins so far identified are glucose-regulated protein 78 (GRP78), protein disulfide isomerase (PDI), galectin-1, and several heat-shock proteins. The goal is to generate a database which emphasizes those proteins most relevant to the use of CHO cells as a host for recombinant protein expression.

Docosapentaenoic acid (C22:5, ?-3) production by Pythium acanthicum

The physiological roles of omega-3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid have been investigated in detail and microbial strains producing these polyunsaturated fatty acids have been characterised. It has recently been suggested that docosapentaenoic acid may have an important role, especially in infant nutrition, and that its positive health effects have been overlooked. Docosapentaenoic acid (C22:5, ω-3) production by a strain of Pythium acanthicum ATCC 18660 was thus investigated. Optimum conditions for growth of P. acanthicum ATCC 18660 and docosapentaenoic acid production were: pH 6.0, temperature 20°C and incubation time, 10 days. Among different saccharides and complex nitrogen sources tested, glucose and sodium glutamate were preferred carbon and nitrogen sources, respectively. Maximum biomass content (10.4 g L−1) and docosapentaenoic acid yield (49.9 mg L−1) were obtained in 10 days. An increase in docosapentaenoic acid volumetric yields to 108–110 mg L−1 was obtained when linseed oil was used to supplement glucose or soy flour-containing medium. Batch feeding of additional glucose or linseed oil further enhanced the docosapentaenoic acid volumetric yield to 132 mg L−1 and 125 mg L−1, respectively, in 14 days. The specific production of docosapentaenoic acid in preliminary experiments ranged from 1.0–5.0 mg g−1 biomass. As conditions were optimised, docosapentaenoic acid specific production titers were generally in the range of 4.0–5.5 mg g−1 and increases in docosapentaenoic acid volumetric production could be attributed to increased biomass production. The limited improvement obtained by modifying culture conditions indicates that increasing volumetric yields of docosapentaenoic acid by modifying culture conditions appears to represent a significant barrier to commercialisation of such a process and suggests a more fundamental manipulation of metabolism and physiology is required.

Epidermal growth factor and temperature regulate keratinocyte differentiation.

The limited life-span and irregularities in epidermal differentiation and barrier function that have restricted the utility of presently available skin culture models for pharmacological and toxicological studies indicate that further modifications of culture conditions are required for optimization of these models. In the present study epidermis reconstructed on de-epidermized dermis was used to investigate the effects of temperature and epidermal growth factor (EGF) on epidermal differentiation and lipogenesis. When cultured at 37 degrees C, keratinocytes formed a well-differentiated epidermis whether EGF was present or not. However, the thickness of the epidermis, particularly of the stratum corneum, was higher in the presence of EGF. Both the differentiation-specific protein markers (keratins 1 and 10, involucrin and transglutaminase) and lipid markers (ceramides) were synthesized. EGF-induced increases in triglyceride content caused accumulation of lipid droplets within the stratum corneum which is indicative of a hyperproliferative effect of EGF. In the absence of EGF, a well-differentiated epidermis was generated at 33 degrees C with a morphology showing a higher resemblance to native epidermis than cultures grown at 37 degrees C. The stratum corneum was less compact and with practically no lipid droplets, irregularly shaped keratohyalin granules were abundant in the stratum granulosum, lamellar body extrusion was improved and the number of stratum corneum layers was reduced to normal levels. However, EGF supplementation had a deleterious effect on epidermal morphogenesis and differentiation of cultures grown at 33 degrees C. The epidermis lacked a stratum granulosum and the stratum corneum contained a high number of nuclear remnants. The synthesis of the early specific protein differentiation markers (keratins 1 and 10) was suppressed on both the protein and mRNA levels without significant interference with the synthesis of late differentiation lipid markers, such as ceramides. From this observation it can be concluded that the synthesis of keratins associated with terminal differentiation is profoundly affected by the presence of EGF and is sensitive to temperature and that of ceramides is not. The finding that TGF alpha did not modulate the morphogenesis and synthesis of keratins 1 and 10 in cultures grown at 33 degrees C indicates possible differences between the postreceptor binding processes of these EGF receptor ligands.

Expression in Escherichia coli of the extracellular basic protease from Dichelobacter nodosus

Dichelobacter nodosus, a Gram-negative obligate anaerobe and the causative agent of ovine footrot, secretes a number of extracellular proteases, one of which is highly basic in nature. The gene (bprV) encoding this basic protease, from virulent strain 198, has been cloned and sequenced. Clone pBR3KB contained the complete bprV gene which constitutively expressed an active protease using its own promoter, when cloned in Escherichia coli. However, levels of protease expression were low and unstable when the clone was expressed in liquid culture. A range of E. coli strains were examined for stable expression; strains NH274 and SURE were found to be better hosts for stable expression than other commonly used E. coli host strains. Stabilization and enhancement of expression was achieved by deletion of the native promoter region and expression from plasmid promoter or promoters, and by modification of culture conditions. The recombinant protease obtained from E. coli was indistinguishable from the native enzyme in size, activity, isoelectric point and immunological properties.

Ciliogenesis in human bronchial epithelial cells cultured at the air-liquid interface.

We present a study on modification of culture conditions in serially cultured human bronchial epithelial cells (HBEC), necessary to achieve bronchial epithelial cells similar to the native epithelium. Cells were obtained from bronchial biopsies and serially cultured using a previously described method (In Vitro Cell. Dev. Biol. 1993; 29A:379-387). At the air-liquid interface, the second and the subsequent passages of HBEC cultures were grown 7 to 31 days, in medium containing fetal calf serum, using de-epidermized dermis or collagen discs as substratum. Scanning and transmission electron microscopy revealed ciliogenesis after 7 days and maturation of the cilia up to 31 days, irrespective of whether de-epidermized dermis or collagen membrane was used. The transmission electron microscopy of the developing cilia showed fibrogranular masses, procentrioles, basal bodies, and in the mature cilia a normal ultrastructure of the axoneme, the nine doublets, the central pair, radial spokes, and dynein arms in the ciliary shaft. In contrast, the submerged cultures showed no signs of ciliogenesis in the same time course. Results of experiments, in which cell seeding density, the substrate used, and the manner of nutrient supplementation were modulated, revealed that the air-exposure of the cultured HBEC is a necessary requirement for the ciliogenesis. The development pathway of ciliated cells in air-exposed HBEC cultures was similar to the differentiation and maturation pattern in human fetal tracheal cells. The in vitro model of human bronchial epithelial cells derived from biopsies obtained by fiberoptic bronchoscopy offers an attractive model for future studies on the function of human bronchial epithelial cells under normal and pathologic conditions.

Enhanced Transformation of Streptococcus mutans by Modifications in Culture Conditions

Enhanced Transformation of Streptococcus mutans by Modifications in Culture Conditions

Modified culture conditions for increased viability and cell wall synthesis in grapevine (Vitis vinifera L. cv. Sultanina) leaf protoplasts

Studies were undertaken to determine optimum conditions for grapevine protoplast culture. Highest viability was obtained at 25° in the dark, and at initial pH values of 5.2 to 7.2, in the presence of 1 or 2% (w/v) sucrose and 0.6 or 0.7 M mannitol or osmolality between 729 and 930 mOsmol kg-1. Optimum plant growth regulators were 6-BAP/NAA at 2.3×10-6/10–15×10-6 M, respectively. 35S-Methionine incorporation into de novo synthesized proteins for protoplasts of Nicotiana tabacum cv Xanthi (a readily regenerating species) and for the recalcitrant grapevine was studied. In tobacco the rate of protein synthesis showed 3 maxima which coincided with the distinct stages of naked protoplasts, cell wall reconstitution and induction of cell division. In grapevine protoplasts the first peak exceeded the second one, whereas no peak corresponding to the induction of cell division was observed.

Effect of epidermal and insulin-like growth factors on vectorial secretion of transferrin by rat Sertoli cells in vitro

Within the seminiferous tubules, the Sertoli cells create an impermeable blood-testis barrier and an unique intratubular microenvironment that fosters the development of spermatozoa. The functional differentiation of spermatozoa therefore requires vectorial secretion by Sertoli cells of substances that cannot cross the blood-testis barrier. We investigated the role of epidermal (EGF) and insulin-like growth factors I and II (IGF-I and IGF-II) in the regulation of vectorial secretion of transferrin by Sertoli cells. In order to study the regulation of vectorial transferrin secretion, we modified culture conditions in the twin chamber culture system to maximise gradients of transferrin secretion. Sertoli cells were plated at high density (3−4 × 10 6 cells/well) into chambers of near equal volume, cultured at 37°C and maintained in simple, fully defined media omitting standard supplements (insulin, EGF, FSH) which affect vectorial transferrin secretion. Using this optimised culture system, maximum gradients of transferrin secretion occurred between days 2 and 3 of culture with preferential secretion (mean ratio 3.7 ± 0.2) directed towards the apical compartment. The transferrin ratio (ratio of transferrin secreted into the upper over the lower chamber) was decreased by insulin and FSH but not by retinoic acid or testosterone, yet all four stimuli increased total transferrin secretion. IGF-I and IGF-II were effective at physiological concentrations (ED 50 = 1 ng/ml) in lowering transferrin ratio and were 100-fold more potent than insulin suggesting that insulin effects on vectorial transferrin secretion by Sertoli cells is mediated through type 1 IGF receptors. EGF also reduced the transferrin ratio (ED 50 = 50 ng/ml) as well as stimulating total transferrin secretion. The hormonally mediated reduction in transferrin ratio was consistently due to enhanced secretion of transferrin into the lower chamber. In the first demonstration of a highly polarised response of Sertoli cells to hormonal stimuli, the effects of insulin, FSH and EGF on vectorial transferrin secretion were effected primarily via the basal membrane of the Sertoli cell and operated independent of mechanisms controlling total transferrin secretion. These results establish a potential role for epidermal and insulin-like growth factors in the paracrine regulation of vectorial secretion by the Sertoli cell, in particular the developmental regulation of vectorial transferrin secretion by Sertoli cells. These findings also indicate that previous studies which included insulin and EGF routinely in culture media have systematically underestimated apically directed transferrin secretion.

Autologous Tumor-Specific Cytotoxicity of Tumor-Infiltrating Lymphocytes Derived from Human Renal Cell Carcinoma

Conditions for generating and expanding cytotoxic tumor-specific, tumor-infiltrating lymphocytes (TIL) were studied to improve the efficacy of adoptive cancer immunotherapy. Thus, we have examined the growth and cytolytic patterns of bulk culture TIL from human renal cell carcinoma (RCC) cultured in low (20 U/ml) or high (1,000 U/ml) dose interleukin (IL)-2, with or without irradiated autologous tumor stimulation. By 55 days in culture, TIL grown in the presence of IL-2 without tumor stimulation lost their lytic activity, whereas those exposed to tumor stimulation maintained high levels of cytotoxicity against autologous and/or nonautologous tumor targets. Only TIL grown with low dose IL-2 and autologous tumor maintained long-term (over 4 months in culture) specific cytotoxicity against the autologous tumor, even upon cryopreservation and regrowth. These TIL were 88-97% and 80% positive for CD3 and CD8, with a persistent subset exhibiting CD4+ CD8+ double positive staining. Their specific cytotoxic activity was major histocompatibility complex Class I-restricted and inhibited by pretreating the TIL with anti-CD3 monoclonal antibody. TIL exposed to the four types of culture conditions, low or high dose IL-2, with or without irradiated autologous tumors, and exhibiting different lytic specificities, all expressed mRNA for interferon-gamma and tumor necrosis factor (TNF)-alpha, but not for IL-1-beta, IL-4, IL-6, and granulocyte-macrophage colony stimulating factor. The degree of TNF-alpha mRNA expression correlated with the degree of autologous tumor-specific cytotoxicity of these TIL. This initial report demonstrates that antigen-specific cytotoxicity against the autologous tumor does, in fact, exist within the RCC tumors.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased permissivity of monkey cells to human adenovirus multiplication is affected by culturing conditions and correlates with both synthesis of virion fiber protein and altered splicing of its mRNA

Human adenoviruses (Ad2) grow poorly in CV-1 (monkey) cells, resulting in an abortive infection. During abortive infections synthesis of the fiber protein is approximately 100-fold depressed compared to its synthesis in CV-1 cells productively infected with a host range mutant of adenovirus (Ad2hr400), while the corresponding mRNA is reduced only 10-fold. We found that passage of CV-1 cells under modified culture conditions resulted in loss of restrictiveness for Ad2 growth. As cells became more permissive, fiber polypeptide synthesis was enhanced although fiber mRNA levels did not necessarily increase. Analysis of the 5′ ends of fiber message isolated from cells in various stages of permissivity showed a direct correlation between efficient synthesis of the fiber protein and an altered splicing pattern of the fiber message.