Optimising fed-batch production of recombinant proteins using the baculovirus expression vector system

Fed-batch culture can offer significant improvement in recombinant protein production compared to batch culture in the baculovirus expression vector system (BEVS), as shown by Nguyen et al. (1993) and Bedard et al. (1994) among others. However, a thorough analysis of fed-batch culture to determine its limits in improving recombinant protein production over batch culture has yet to be performed. In this work, this issue is addressed by the optimisation of single-addition fed-batch culture. This type of fed-batch culture involves the manual addition of a multi-component nutrient feed to batch culture before infection with the baculovirus. The nutrient feed consists of yeastolate ultrafiltrate, lipids, amino acids, vitamins, trace elements, and glucose, which were added to batch cultures of Spodoptera frugiperda (Sf9) cells before infection with a recombinant Autographa californica nuclear polyhedrosis virus (Ac-NPV) expressing beta-galactosidase (beta-Gal). The fed-batch production of beta-Gal was optimised using response surface methods (RSM). The optimisation was performed in two stages, starting with a screening procedure to determine the most important variables and ending with a central-composite experiment to obtain a response surface model of volumetric beta-Gal production. The predicted optimum volumetric yield of beta-Gal in fed-batch culture was 2.4-fold that of the best yields in batch culture. This result was confirmed by a statistical analysis of the best fed-batch and batch data (with average beta-Gal yields of 1.2 and 0.5 g/L, respectively) obtained from this laboratory. The response surface model generated can be used to design a more economical fed-batch operation, in which nutrient feed volumes are minimised while maintaining acceptable improvements in beta-Gal yield. (C) 1998 John Wiley & Sons, Inc.

Th2-type CD4(+) cells neither enhance nor suppress antitumor CTL activity in a mouse tumor model

Many cervical cancers express the E7 protein of human papillomavirus 16 as a tumor-specific Ag (TSA). To establish the role of E7-specific T cell help in CD8(+) CTL-mediated tumor regression, C57BL/6J mice were immunized with E7 protein or with a peptide (GF001) comprising a minimal CTL epitope of E7, together with different adjuvants, Immunized mice were challenged with an E7-expressing tumor cell line, EL4.E7. Growth of EL4.E7 was reduced following immunization with E7 and Quil-A (an adjuvant that induced a Th1-type response to E7) or with GF001 and Quil-A, Depletion of CD8(+) cells, but not CD4(+) cells, from an immunized animal abrogated protection, confirming that E7-specific CTL are necessary and sufficient for TSA-specific protection in this model. Immunization with E7 and Algammulin (an alum-based adjuvant) induced a Th2-like response and provided; no tumor protection. To investigate whether a Th2 T helper response to E7 could prevent the development of an E7-specific CTL-mediated protection, mice were simultaneously immunized with E7/Algammulin and GF001/Quil-A or, alternatively, were immunized with GF011/Quil-A 8 wk after immunization with E7/Algammulin, Tumor protection was observed in each case. We conclude that an established Th2 response to a TSA does not prevent the development of TSA-specific tumor protective CTL.

Split tolerance to a viral antigen expressed in thymic epithelium and keratinocytes

When expressed as a transgene from the keratin 14 (K14) promoter in an MHC class II-deficient mouse, I-Ab expressed in thymic cortical epithelium promotes positive but not negative selection of I-Ab-restricted CD4(+) T cells (Laufer, T. M. et al., Nature 1996. 383:81-85). Transgenic mice expressing the E7 protein of human papilloma virus 16 from the K14 promoter were studied to determine the consequence of expression of a cytoplasmic/nuclear protein from the K14 promoter. K14E7-transgenic mice express E7 in the thymus and skin without evidence for autoimmunity to E7. Repeated immunization of FVB(H-2(q)) or F1(C57BV6JxFVB) mice with E7 elicited similar antibody responses to the defined B cell epitopes of E7 in K14E7-transgenic and non-transgenic animals. In contrast, for each genetic background, a single immunization with E7 elicited demonstrable T cell proliferative responses to the major promiscuous T helper epitope of E7 in the transgenic but not the non-transgenic animals. Further,E7-immunized non-transgenic F1 (FVBxC57BL/6J) animals developed strong E7-specific cytotoxic T lymphocyte (CTL) responses and were protected against challenge with E7(+) tumors, whereas similarly immunized K14E7-transgenic animals had a markedly reduced CTL response to E7 and no E7-specific tumor protection was observed, although the antibody and CTL response to ovalbumin was normal. Expression of E7 protein as a transgene from the K14 promoter in the skin and thymus thus induces E7-specific tolerance in the cytotoxic T effector repertoire, together with expansion of the E7-specific T helper repertoire. These findings demonstrate that limited tissue distribution of an autoantigen may result in split tolerance to that autoantigen.

Environmental poisoning: Presentation and management

Environmental poisoning is most commonly associated with chronic longterm exposure to toxins rather than to acute exposure. Such repeated exposure to sublethal doses of compounds and elements presents problems in risk assessment. This is primarily because the data are unavailable to describe relationships between dose and effect at lower levels of exposure to toxins. Bioavailability of toxins also presents a problem because the data on bioavailability are sparse and seldom as high as the default of 100% bioavailability commonly used in risk assessment. Examples are presented of two toxins: arsenic as an elemental anthropogenic and geologic poison and ciguatoxin, a polyether ladder compound, as a toxin produced naturally by dinoflagellates. Bioavailability drives the toxicity of arsenic from contaminated sites, whereas tissue accumulation drives the toxicity of ciguatoxin. Considerable benefit is derived from the harmonization of regulatory processes where there is linkage of health and environmental factors in the derivation of credible risk assessment.

Molecular development of the olfactory nerve pathway

There are, at least, two major questions concerning the molecular development of the olfactory nerve pathway. First, what are the molecular cues responsible for guiding axons from the nasal cavity to the olfactory bulb? Second, what is the molecular basis of axon targeting to specific glomeruli once axons reach the olfactory bulb? Studies in the primary olfactory pathway have focused on the role of the extracellular matrix and ensheathing cells in establishing an initial substrate for growth of pioneer axons between the periphery and brain. The primary axons also express a multitude of cell adhesion molecules that regulate fasciculation of axons and hence may play a role in fascicle formation in the olfactory nerve. Although the olfactory neuroepithelium principally consists of a morphologically homogeneous class of primary olfactory neurons, there are numerous subpopulations of olfactory neurons expressing chemically distinct phenotypes. In particular, numerous subpopulations have been characterized by expression of unique carbohydrate residues and olfactory receptor proteins. Some of these molecules have recently been implicated in axon guidance and targeting to specific glomeruli.

A cell type-specific constitutive point mutant of the common beta-subunit of the human granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin (IL)-3, and IL-5 receptors requires the GM-CSF receptor alpha-subunit for activation

The high affinity receptor for human granulocyte-macrophage colony-stimulating factor (GM-CSF) consists of a cytokine-specific alpha-subunit (hGMR alpha) and a common signal-transducing beta-subunit (hpc) that is shared with the interleukin-3 and -5 receptors, We have previously identified a constitutively active extracellular point mutant of hpc, I374N, that can confer factor independence on murine FDC-P1 cells but not BAF-B03 or CTLL-2 cells (Jenkins, B. J., D'Andrea, R. J., and Gonda, T. J. (1995) EMBO J. 14, 4276-4287), This restricted activity suggested the involvement of cell type-specific signaling molecules in the activation of this mutant. We report here that one such molecule is the mouse GMR alpha (mGMR alpha) subunit, since introduction of mGMR alpha, but not hGMR alpha, into BAF-B03 or CTLL-2 cells expressing the I374N mutant conferred factor independence, Experiments utilizing mouse/human chimeric GMR alpha subunits indicated that the species specificity lies in the extracellular domain of GMRa. Importantly, the requirement for mGMR alpha correlated with the ability of I374N (but not wild-type hpc) to constitutively associate with mGMRa. Expression of I374N in human factor-dependent UT7 cells also led to factor-independent proliferation, with concomitant up-regulation of hGMR alpha surface expression. Taken together, these findings suggest a critical role for association with GMR alpha in the constitutive activity of I374N.

Hepatocellular carcinoma

Hepatitis C infection is associated with the development of hepatocellular carcinoma, and progress has been made in a number of areas. Transgenic mice lines expressing the hepatitis C core protein develop hepatic steatosis, adenomas, and hepatocellular carcinomas, with no significant hepatitis or fibrosis. This implies that hepatitis C can lead directly to malignant transformation, A new lesion, irregular regeneration, has been described in chronic hepatitis C infection and is associated with a 15-fold increase in the relative risk of developing hepatocellular carcinoma. A minority of patients with hepatitis C-related hepatocellular carcinoma have intense lymphocytic infiltration of the cancer, a feature associated with a better prognosis, Several studies have confirmed the association between large cell dysplasia and hepatocellular carcinoma, However, large cell dysplasia may not be a premalignant lesion and instead may be a marker for premalignant alterations elsewhere in the liver. Oral contraceptives previously have been linked to an increased risk of hepatocellular carcinoma. A large multicenter European case-control study has shown minimal increased risk of hepatocellular carcinoma with use of steroidal contraception. Tamoxifen had shown promise in the management of advanced hepatocellular carcinoma. However, a randomized placebo-controlled study of 477 patients with hepatocellular carcinoma found no benefit from tamoxifen, In a preliminary study, however, octreotide has shown improved survival and quality of life in patients with advanced hepatocellular carcinoma, Finally, interferon treatment continues to be linked to a reduced risk of hepatocellular carcinoma in patients with hepatitis C, These studies generally are not randomized, and a randomized prospective study is required to address this issue. (C) 1999 Lippincott Williams & Wilkins, Inc.

Novel guidance cues during neuronal pathfinding in the early scaffold of axon tracts in the rostral brain

A scaffold of axons consisting of a pair of longitudinal tracts and several commissures is established during early development of the vertebrate brain. We report here that NOC-2, a cell surface carbohydrate, is selectively expressed by a subpopulation of growing axons in this scaffold in Xenopus. NOC-2 is present on two glycoproteins, one of which is a novel glycoform of the neural cell adhesion molecule N-CAM. When the function of NOC-2 was perturbed using either soluble carbohydrates or anti-NOC-2 antibodies, axons expressing NOC-2 exhibited aberrant growth at specific points in their pathway. NOC-2 is the first-identified axon guidance molecule essential for development of the axon scaffold in the embryonic vertebrate brain.

Distribution of interleukin-2,-4,-10, tumour necrosis factor-alpha and transforming growth factor-beta mRNAs in oral lichen planus

In the present study. MRNA for the cytokines interleukin-2 (IL-2), IL-4, IL-10 tumour necrosis factor-alpha (TNF-alpha) and transforming growth factor beta-1 (TGF-beta-1) were investigated in oral lichen planus (OLP) lesions using in situ hybridization with S-35-labelled oligonucleotide probes on frozen tissue sections. In addition, the expression of interferon-gamma (IFN-gamma), IL-10 and IL-4 mRNAs was analysed in cultured lesional T lymphocytes from oral lichen planus by polymerase chain reaction. Cells expressing mRNA for IL-2, IL-4, IL-10, TNF-alpha and TGF-beta(1) were found in all the biopsies studied. Approximately 1-2% of the total number of infiltrating cells in the lesions were positive for each of the different cytokine mRNAs. Most biopsies contained basement membrane-oriented, mRNA-positive cells. In the cultured T-cell lines, message for IFN-gamma was detected in all the patients, IL-10 in all but one, and IL-4 in just one of the seven patients investigated. The results suggest that mRNA for both pro- and anti-inflammatory cytokines, i.e., mixed T-helper 1 (T(H)1) and T(H)2 cytokine profiles, are generated simultaneously by a limited number of cells in chronic lesions of OLP. (C) 1999 Elsevier Science Ltd. All rights reserved.

Cytomegalovirus evasion of natural killer cell responses

Natural killer (NK) cells are an important component of the innate cellular immune system. They are particularly important during the early immune responses following virus infection, prior to the induction of cytotoxic T cells (CTL). Unlike CTL, which recognize specific peptides displayed on the surface of cells by class I MHC, NK cells respond to aberrant expression of cell surface molecules, in particular class I MHC, in a non-specific manner. Thus, cells expressing low levels of surface class I MHC are susceptible to recognition by NK cells, with concomitant triggering of cytolytic and cytokine-mediated responses. Many viruses, including the cytomegaloviruses, downregulate cell surface MHC class I: this is likely to provide protection against CTL-mediated clearance of infected cells, but may also render infected cells sensitive to NK-cell attack. This review focuses upon cytomegalovirus-encoded proteins that are believed to promote evasion of NK-cell-mediated immunity. The class I MHC homologues, encoded by all cytomegaloviruses characterised to date, have been implicated as molecular 'decoys', which may mimic the ability of cellular MHC class I to inhibit NK-cell functions. Results from studies in vitro are not uniform, but in general they support the proposal that the class I homologues engage inhibitory receptors from NK cells and other cell types that normally interact with cellular class I. Consistent with this, in vivo studies of murine cytomegalovirus indicate that the class I homologue is required for efficient evasion of NK-cell-mediated clearance. Recently a second murine cytomegalovirus protein, a C-C chemokine homologue, has been implicated as promoting evasion of NK and T-cell-mediated clearance in vivo.

Structure-activity relationships of omega-conotoxins MVIIA, MVIIC and 14 loop splice hybrids at N and P/Q-type calcium channels

The omega-conotoxins are a set of structurally related, four-loop, six cysteine containing peptides, that have a range of selectivities for different subtypes of the voltage-sensitive calcium channel (VSCC). To investigate the basis of the selectivity displayed by these peptides, we have studied the binding affinities of two naturally occurring omega-conotoxins, MVIIA and MVIIC and a series of 14 MVIIA/MVIIC loop hybrids using radioligand binding assays for N and P/Q-type Ca2+ channels in rat brain tissue. A selectivity profile was developed from the ratio of relative potencies at N-type VSCCs (using [I-125]GVIA radioligand binding assays) and P/Q-type VSCCs (using [I-125]MVIIC radioligand binding assays). in these peptides, loops 2 and 4 make the greatest contribution to VSCC subtype selectivity, while the effects of loops 1 and 3 are negligible. Peptides with homogenous combinations of loop 2 and 4 display clear selectivity preferences, while those with heterogeneous combinations of loops 2 and 4 are less discriminatory. H-1 NMR spectroscopy revealed that the global folds of MVIIA, MVIIC and the 14 loop hybrid peptides were similar; however, several differences in local structure were identified. Based on the binding data and the 3D structures of MVIIA, GVIA and MVIIC, we have developed a preliminary pharmacophore based on the omega-conotoxin residues most Likely to interact with the N-type VSCC. (C) 1999 Academic Press.

Engineered detoxification confers resistance against a pathogenic bacterium

We generated transgenic sugarcane plants that express an albicidin detoxifying gene (albD), which was cloned from a bacterium that provides biocontrol against leaf scald disease. Plants with albicidin detoxification capacity equivalent to 1-10 ng of AlbD enzyme per mg of leaf protein did not develop chlorotic disease symptoms in inoculated leaves, whereas all untransformed control plants developed severe symptoms. Transgenic lines with high AlbD activity in young stems were also protected against systemic multiplication of the pathogen, which is the precursor to economic disease. We have shown that genetic modification to express a toxin-resistance gene can confer resistance to both disease symptoms and multiplication of a toxigenic pathogen in its host.

MiAMP1, a novel protein from Macadamia integrifolia adopts a Greek key beta-barrel fold unique amongst plant antimicrobial proteins

MiAMP1 is a recently discovered 76 amino acid residue, highly basic protein from the nut kernel of:Macadamia integrifolia which possesses no sequence homology to any known protein and inhibits the growth of several microbial plant pathogens in vitro while having no effect on mammalian or plant cells. It is considered to be a potentially useful tool for the genetic engineering of disease resistance in transgenic crop plants and for the design of new fungicides. The three-dimensional structure of MiAMP1 was determined through homonuclear and heteronuclear (N-15) 2D NMR spectroscopy and subsequent simulated annealing calculations with the ultimate aim of understanding the structure-activity relationships of the protein. MiAMP1 is made up of eight beta-strands which are arranged in two Greek key motifs. These Greek key motifs associate to form a Greek key beta-barrel. This structure is unique amongst plant antimicrobial proteins and forms a new class which we term the beta-barrelins. Interestingly, the structure of MiAMP1 bears remarkable similarity to a yeast killer toxin from Williopsis mrakii. This toxin acts by inhibiting beta-glucan synthesis and thereby cell wall construction in sensitive strains of yeast. The structural similarity of MiAMP1 and WmKT, which originate from plant and fungal phyla respectively, may reflect a similar mode of action. (C) 1999 Academic Press.

Development of P2 olfactory glomeruli in P2-internal ribosome entry site-tau-lacZ transgenic mice

Primary olfactory neurons project their axons to the olfactory bulb, where they terminate in discrete loci called glomeruli. All neurons expressing the same odorant receptor appear to terminate in a few glomeruli in each olfactory bulb. In the P2-IRES-tau-LacZ line of transgenic mice, LacZ is expressed in the perikarya and axons of primary olfactory neurons that express the P2 odorant receptor. In the present study, we examined the developmental appearance of P2 neurons, the topographical targeting of P2 axons, as well as the formation of P2 glomeruli in the olfactory bulb. P2 axons were first detected in the olfactory nerve fiber layer at embryonic day 14.5 (E14.5), and by E15.5 these axons terminated in a broad locus in the presumptive glomerular layer. During the next 5 embryonic days, the elongated cluster of axons developed into discrete glomerulus-like structures. In many cases, glomeruli appeared as pairs, which were initially connected by a fascicle of P2 axons. This connection was lost by postnatal day 7.5, and double glomeruli at the same locus were observed in 85% of adult animals. During the early postnatal period, there was considerable mistargeting of P2 axons. In some cases P2 axons entered inappropriate glomeruli or continued to grow past the glomerular layer into the deeper layers of the olfactory bulb. These aberrant axons were not observed in adult animals. These results indicate that olfactory axons exhibit errors while converging onto a specific glomerulus and suggest that guidance cues may be diffusely distributed at target sites in the olfactory bulb.

Altered adhesion, proliferation and death in neural cultures from adults with schizophrenia

The causes of schizophrenia are unknown, but there is evidence linking subtle deviations in neural development with schizophrenia. Embryonic brain development cannot be studied in an adult with schizophrenia, but neurogenesis and early events in neuronal differentiation can be investigated throughout adult life in the human olfactory epithelium. Our past research has demonstrated that neuronal cultures can be derived from biopsy of the human adult olfactory epithelium. In the present study, we examined mechanisms related to neurogenesis and neuronal differentiation in adults with schizophrenia versus well controls. Forty biopsies were collected under local anaesthesia from ten individuals with DSM III-R schizophrenia and ten age- and sex-matched well controls. All patients, except one, were receiving antipsychotic medication at the time of the biopsy, Immunostaining for neuronal markers indicated that neurogenesis occurred in the biopsies from both patients and controls since all contained cells expressing tubulin and/or olfactory marker protein. The major findings of this study are: 1. biopsies from patients with schizophrenia showed a significantly reduced ability to attach to the culture slide: 29.9% of patient biopsies attached compared to 73.5% of control biopsies; 2. biopsies from patients with schizophrenia had a significantly greater proportion of cells undergoing mitosis: 0.69% in the patients compared to 0.29% in the controls; and 3. dopamine (10 mu M) significantly increased the proportion of apoptotic cells in the control cultures but significantly decreased the proportion in patients' cultures. (C) 1999 Elsevier Science B.V. All rights reserved.