Treatment with the TGF-b superfamily cytokine MIC-1/GDF15 reduces the adiposity and corrects the metabolic dysfunction of mice with diet-induced obesity.

OBJECTIVES: To test the potential efficacy of recombinant macrophage inhibitory cytokine-1 (MIC-1/GDF15) as an obesity therapeutic. METHODS: Male C57BL/6 J mice, either fed on normal chow or high-fat diet for 16 weeks to induce diet-induced obesity, were infused with either recombinant MIC-1/GDF15 or vehicle for 34 days by osmotic minipump. During the experimental period metabolic parameters were measured. Blood and tissue were collected for analysis of inflammatory markers. RESULTS: MIC-1/GDF15 decreased food intake and body weight of high-fat-fed and chow-fed mice compared with their vehicle-treated control mice. MIC-1/GDF15 reduced body weight, accompanied by greater reduction in fat mass in high-fat-fed mice compared to its effect on chow-fed mice. Further, whilst MIC-1/GDF15-treated chow-fed mice lost lean as well as fat mass, MIC-1/GDF15-treated high-fat-fed mice lost fat mass alone. This reduction in body weight and adiposity was due largely to reduced food intake, but MIC-1/GDF15-treated high-fat-fed mice also displayed increased energy expenditure that may be due to increased thermogenesis. MIC-1/GDF15-treated high-fat-fed mice also had higher circulating level of adiponectin and lower tissue expression, and circulating levels of leptin and inflammatory mediators associated with insulin resistance. Peripheral insulin and glucose intolerance were improved in both MIC-1/GDF15-treated high-fat-fed and chow-fed mice compared to that of their vehicle-treated control mice. CONCLUSIONS: MIC-1/GDF15 is highly effective in reducing adiposity and correcting the metabolic dysfunction of mice with high-fat fed. These studies suggest that MIC-1/GDF15 may be a candidate anti-obesity therapeutic.

Macrophage inhibitory cytokine-1/growth differentiation factor-15 as a predictor of colonic neoplasia.

BACKGROUND: Serum macrophage inhibitory cytokine-1 (MIC-1/GDF15) concentration has been associated with colonic adenomas and carcinoma. AIMS: To determine whether circulating MIC-1/GDF15 serum concentrations are higher in the presence of adenomas and whether the level decreases after excision. METHODS: Patients were recruited prospectively from a single centre and stratified into five groups: no polyps (NP); hyperplastic polyps (HP); sessile serrated ademona (SSA); adenomas (AP); and colorectal carcinoma (CRC). Blood samples were collected immediately before and 4 weeks after colonoscopy. MIC-1/GDF15 serum levels were quantified using ELISA. RESULTS: Participants (n=301) were stratified as: NP; n=116 (52%), HP; n=37 (12%), SSA; n=19 (7%), AP; n=68 (23%); and CRC; n=3 (1%). Patients were excluded from the study due to nondiagnostic pathology (n=9, 3%) and exclusion criteria (n=20, 6%). In the 272 remaining subjects (M=149; F=123), age (P=.005), history of colonic polyps (P=.003) and family history of colonic polyps (P=.002) were associated with presence of adenomas. Baseline median MIC-1/GDF15 serum levels increased significantly from NP 609 (460-797) pg/mL, HP 582 (466-852) pg/mL, SSA 561 (446-837) pg/mL to AP 723 (602-1122) pg/mL and CRC 1107 (897-1107) pg/mL; (P<.001). In the pre- and postpolypectomy paired adenoma samples median MIC-1/GDF15 reduced significantly from 722 (603-1164) pg/mL to 685 (561-944) pg/mL (P=.002). A ROC analysis for serum MIC-1/GDF15 to identify adenomatous polyps indicated an area under the curve of 0.71. CONCLUSIONS: Our data suggest that serum MIC-1/GDF15 has the diagnostic characteristics to increase the detection of colonic neoplasia and improve screening.

Gene expression profiling of Arabidopsis meiocytes.

Meiosis is a special type of cell division present in all organisms that reproduce by sexual reproduction. It ensures the transition between the sporophytic and gametophytic state and allows gamete production through meiotic recombination and chromosome number reduction. In this paper, we describe a technique for the isolation of Arabidopsis thaliana male meiocytes. From this cellular material, it was then possible to develop large-scale transcriptome studies using CATMA microarrays and thus to obtain an overview of genes expressed during Arabidopsis meiosis. The expression profiles were studied with either stringent statistical criteria or by performing clustering. Both methods resulted in gene clusters enriched in meiosis-specific genes (from 14- to 55-fold). Analysis of these data provided a unique set of genes that will be pivotal to further analysis aimed at understanding the meiotic process.

A pilot study to assess an online training module to quickly identify drugs on resuscitation trays.

OBJECTIVE: To measure the median time required for healthcare professionals to identify drugs on resuscitation trays and to assess the usefulness of an online training module used to identify drugs on resuscitation trays. STUDY DESIGN: This is a descriptive pilot study conducted in a mother-child university hospital center with a convenience sample of physicians, residents, nurses, pharmacists, pharmacy students or pharmacy technicians (10/group). METHOD: Participants were given a questionnaire before and after using a simulation module to identify drugs on resuscitation drug trays (30 questions on the full trays with 43 drugs and 15 questions on the partial trays with 21 drugs). The identification times were measured for each drug and for each tray. RESULTS: The median time to identify the drugs varied from 4s (range 2-46) for dextrose to 18s (range 4-78) for epinephrine. The median times to locate a drug on full and partial trays were, respectively: pharmacists 7 (2-103) and 6 (1-31), physicians 10 (3-78) and 7 (2-61) and nurses 10 (3-83) and 7 (2-53). All 60 participants strongly agreed that the online simulation module was a good tool for healthcare staff and that it would allow them to locate drugs more quickly in emergency situations. CONCLUSION: The online simulation module can be used by various groups of professionals and it can allow them to locate drugs on resuscitation tray more rapidly during an emergency.

Evidence of heterogeneity in the antibody response against the platelet antigen 3a; recognition of an 11-mer peptide carrying the HPA-3a polymorphic determinant.

BACKGROUND: The immune processes involved in the development of alloantibodies against the human platelet antigens in alloimmune disorders remain unclear. Antibody recognition of the platelet antigens on their respective platelet glycoproteins has been shown to be dependent on glycoprotein conformation. Furthermore, the post-translational modification of glycoproteins adds complexity to the alloantigenic determinants. METHODS: Nine anti-HPA-3a sera along with several control sera were tested for reactivity to an 11-mer peptide straddling the HPA-3a/b polymorphism. Sera found to specifically recognize the 3a peptide were further assessed by platelet pre-exposure and immunoblotting. RESULTS: Three of the nine antisera were found to specifically recognize an 11-mer synthetic 3a peptide by ELISA. Further analysis of all anti-HPA-3a sera by Western blot showed that only those reactive to the 3a peptide were able to bind both reduced and non-reduced GPIIb. CONCLUSION: The results presented in this study provide the first known evidence for the identification of an antibody population capable of recognizing a linear and non-glycosylated form of the HPA-3a epitope.

Golgi apparatus studied in vitreous sections.

Cryo-electron microscopy of vitrified specimen is the method of choice to explore cellular ultrastructure at high resolution as close as possible to the native state and environment. In this study, we investigated the Golgi apparatus - the main organelle of the secretory pathway. Cultured mammalian cells were fixed by high-pressure freezing, sectioned in vitreous ice and subjected to cryo-electron microscopy and cryo-electron tomography. Although the overall morphology of Golgi stacks was comparable to well prepared and plastic-embedded samples, in detail we reached much higher resolution in terms of distinction between biological structures based on their native density. On cisternal buds and peri-Golgi vesicles--some associated with microtubules--we detected two different subtypes of COPI coats: (1) a homogenous coat and (2) an inhomogeneous spiky coat, providing an 8-9 nm regularity, clearly distinct from clathrin coat. Next, we monitored the secretion of cargo, namely, procollagen I, through the Golgi complex. Temporally correlated with fluorescence microscopy, we performed three-dimensional cryo-electron tomography analysis and detected Golgi cisternae enlarged to saccules, containing cargo and showing inter-cisternal connections. Our work provides a first step towards the high-resolution description of the secretory pathway in native vitrified samples and describes the challenges associated with this attempt.

Cryoelectron microscopy of vitrified sections: a new challenge for the analysis of functional nuclear architecture.

Cryoelectron microscopy of vitrified sections has become a powerful tool for investigating the fine structural features of cellular compartments. In the present study, this approach has been applied in order to explore the ultrastructural morphology of the interphase nucleus in different mammalian cultured cells. Rat hepatoma, Chinese hamster ovary and Potorus kidney cells were cryofixed by high-pressure freezing and the cryosections were examined at low temperature by transmission electron microscopy. Our results show that while the contrast of nuclear structural domains is remarkably homogeneous in hydrated sections, some of them can be recognised due to their characteristic texture. Thus, condensed chromatin appears finely granular and the perichromatin region contains rather abundant fibro-granular elements suggesting the presence of dispersed chromatin fibres and of perichromatin fibrils and granules. The interchromatin space looks homogeneous and interchromatin granules have not been identified under these preparative conditions. In the nucleolus, the most striking feature is the granular component, while the other parts of the nucleolar body, which appear less contrasted, are difficult to resolve. The nuclear envelope is easily recognisable with its regular perinuclear space and nuclear pore complexes. Our observations are discussed in the context of results obtained by other, more conventional electron microscopic methods.

Cereulide, the emetic toxin of Bacillus cereus, is putatively a product of nonribosomal peptide synthesis.

AIMS: To determine if cereulide, the emetic toxin produced by Bacillus cereus, is produced by a nonribosomal peptide synthetase (NRPS). METHODS AND RESULTS: NC Y, an emetic strain of Bacillus cereus, was examined for a NRPS gene using PCR with primers recognizing a fragment of a NRPS gene from the cyanobacterium Microcystis. The amplicon was sequenced and compared with other gene sequences using BLAST analysis, which showed that the amplicon from strain NC Y was similar in sequence to peptide synthetase genes in other micro-organisms, including Bacillus subtilis and B. brevis, while no such sequence was found in the complete genome sequence of a nonemetic strain of B. cereus. Specific PCR primers were then designed and used to screen 40 B. cereus isolates previously implicated in outbreaks of foodborne illness. The isolates were also screened for toxin production using the MTT cell cytotoxicity assay. PCR and MTT assay screening of the B. cereus isolates revealed a high correlation between the presence of the NRPS gene and cereulide production. CONCLUSIONS: The results indicate that cereulide is produced by a NRPS complex. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first study to provide evidence identifying the mechanism of production of cereulide, the emetic toxin of B. cereus. The PCR primers developed in the study allow determination of the potential for cereulide production among isolates of B. cereus.

Finger dominance.

Two hundred and ninety-eight normal participants were reviewed to assess finger dominance. Participants were recruited from several large hospitals across the north-west of England and the study was done under blind conditions. From the 298 people originally in the trial, six had to be excluded as the dominant finger could not be confirmed with certainty. The index finger was the most dominant in 82.5% of the test group. However, in 10.6% of the test group, the middle finger was found to be the most dominant.

Cloning and expression of human V-genes derived from phage display libraries as fully assembled human anti-TNF alpha monoclonal antibodies.

BACKGROUND: With the advent of phage antibody libraries, access to completely human antibody fragments is feasible, either by direct selection from human antibody libraries, or by guided selection. After selection, Fabs and scFvs may need to be expressed as complete antibodies in mammalian cells for further characterisation, or if effector functions are required. OBJECTIVES: To rebuild and express the human anti-TNF alpha antibody Fab-P3A2 (isolated as a Fab fragment from phage display libraries by guided selection) as a fully assembled, functional human antibody (gamma-1, lambda) in Sp2/0 myeloma cells, and to perform preliminary characterisation studies of the secreted IgG1 molecule. A further objective was to investigate the kinetics of human antibody production and the stability of antibody secretion in transfectomas cultured in various media formulations. STUDY DESIGN: A tripartite strategy was employed for cloning heavy chain gene (VH)-P3 and light chain gene V lambda-A2-C lambda into mammalian cell expression vectors p alpha Lys-30 and p alpha Lys-17 respectively. The cell line P3A2.B5 was isolated after co-transfection of Sp2/0 mouse myelomas with the constructs, expanded and weaned into a protein free medium. Fully assembled Ig-P3A2 antibody was purified by Protein A affinity chromatography and characterised with respect to size of antibody chains, and affinity for human TNF alpha. Stability of secretion was investigated by extended serial sub-culture and analysis of P3A2.B5 sub-clones. Strategies of media enrichment were tested for any effect on antibody productivity by selected P3A2.B5 sub-clones. RESULTS: The cell line P3A2.B5 secreted an assembled, human antibody Ig-P3A2, with heavy and light chains of molecular weight 55 and 28 KD respectively. Equilibrium capture studies showed Ig-P3A2 to have a dissociation constant of approximately 1.5 x 10(-8) M. The mean specific productivity of the cell line increased from 1.2 pg/cell/day to 7.8 pg/cell/day by a combination of medium enrichment and serum reduction. Prolonged serial sub-culture of P3A2.B5 showed the cell line to be unstable with respect to antibody secretion. CONCLUSIONS: We have outlined a method for expression of human V genes as assembled antibodies in Sp2/0 myeloma cells. A cloning strategy for the stable expression of scFv or Fab genes isolated from phage display libraries as assembled human antibodies of the IgGl subclass in Sp2/0 myeloma cells has been described. For maximising specific productivity of antibody-producing cell lines, supplementation of culture media with glucose, glutamine and amino acids increases antibody yield significantly compared to that in conventional media, indicating the latter is stoichiometrically limiting for production purposes.

Amino acid metabolism during batch culture of a murine hybridoma, AFP-27.

This paper presents batch culture data of the murine hybridoma, AFP-27, cultured in conventional basal media and in a nutrient-rich modified version. Expression of antibody was fivefold higher in the enriched formulation, with significant product secretion in the decline phase. Cultures were initiated at conventional inculation densities (1 ∼ 2 × 10(5) viable cells ml(-1)) and high inoculation densities (1.5 ∼ 1.7 × 10(6) viable cells ml(-1)). Amino acid levels have been reported for all cultures, with apparent differences described. Relative levels of intracellular amino acids are also reported, with significant accumulation of proline, glycine and alanine. The results have significance in the design of enriched media which are clearly beneficial for commercial production of antibodies from hybridomas.

Carbohydrate and amino acid metabolism during batch culture of a human lymphoblastoid cell line, BTSN6.

This work presents data on the carbohydrate and amino acid metabolism of a lymphoblastoid cell line producing an IgG1 antibody. In static culture, it was observed that lactate levels were significantly lowered when the cells were cultured on galactose as a carbon source. The use of carbohydrate substitution may be useful in lowering lactate levels, if it is established that this component is toxic to the cells. In addition, carbohydrate substitution may be used to modify glycosylation patterns and hence pharmacokinetic properties of glycoproteins.The amino acids glutamine and tryptophan were shown to be limiting in batch culture on this medium (DR, a 1:1 mixture of DMEM and RPMI, with 4mM glutamine). Amino acids produced included alanine, proline and glutamate. Serine was consumed to exhaustion, which was followed by a depletion of extracellular glycine. Amino acid metabolism, specific antibody productivity and specific growth rate were shown to be functions of the inoculation density in stirred flask culture. The results have implications for the design of media for both low and high density antibody manufacture by these cell lines.

Biosynthesis of protein products by animal cells. Are growth and non-growth associated concepts valid or useful?

The application of simple growth and non-growth associated concepts from microbial systems describing substrate uptake and production formation is considered unlikely to assist in the understanding of antibody formation and, hence, in maximising antibody yield. Such concepts have many significant limitations - notably, their strict application only to products of catabolic pathways and their inability to include metabolisms which either have multiple catabolic pathways (eg, fermentation and respiration in yeast and animal cells) or in which the major product of interest is predominantly anabolic in nature (eg. amino acid production in bacteria and antibody formation in animal cells). In addition, products which undergo an assembly and secretion process or a secretion process which allows intracellular pools of product to exist are also not well described by such simple relationships. In this work, inadequacies in the current approach to the study of the kinetics of growth of hybridoma cells and antibody production are described and the examples of growth ofSaccharomyces cerevisiae andCandida utilis, amino acid production by bacteria and antibody production by animal cells are used to illustrate these limitations. Having identified these limitations, suggestions are made as to how studies might be undertaken to assist our future understanding of the process of antibody manufacture and, subsequently, maximizing antibody yield. The process of characterising the metabolism of anabolic products is subject to detailed computer simulation of the pathways involved. It is argued that such approaches will assist us in understanding more fully the nature of biosynthetic products and how they integrate with the major energy producing pathways of the cell and the cell cycle. This will assist in maximising the yield of such products.

Dynamic structure of Agrobacterium tumefaciens Ti plasmids.

Agrobacterium tumefaciens C58F is a variant of strain C58 which generates a high proportion of avirulent mutants in the presence of the virulence (vir) gene inducer acetosyringone. These mutants are altered in the Ti plasmid and do not respond to the acetosyringone signal (C. Fortin, E. W. Nester, and P. Dion, J. Bacteriol. 174:5676-5685, 1992). The physical organization of the Ti plasmid was compared in strain C58 and its variant. One feature distinguishing pTiC58F from its parent plasmid was the presence of the insertion element IS426. Three copies of this element were detected in the strain C58 chromosome, whereas two additional copies were found in strain C58F, including one copy in the Ti plasmid. This particular copy of IS426 was associated with the region of arginine and nopaline catabolism of pTiC58F. Most of the avirulent mutants recovered following growth of strain C58F in the presence of acetosyringone were complemented by clones carrying either virA or virG. Element IS426 was no longer found in the arginine and nopaline catabolism region of the Ti plasmids from the virA and virG mutants, but it resided in the particular KpnI fragment containing the modified vir locus. Behavior of a strain C58F derivative, which was inactivated in a chromosomal component required for the response to acetosyringone, was consistent with the possibility that vir gene induction is essential to the massive production of avirulent mutants.

An analysis of some batch and continuous kinetic data of specific monoclonal antibody production from hybridomas.

An analysis of batch and continuous kinetic data obtained from hybridoma cell cultures has been performed with particular reference to the existence of specific antibody production profiles. The results presented by several groups, including our own, have been studied. Our analysis suggests that different interpretations of the data can be made to those previously presented in the literature. In view of the significance of these profiles, particularly in terms of production strategies designed to maximise antibody production, we believe that more consideration needs to be given to accuracy in reporting of kinetic studies in the future.

A comparison of different culture methods for hybridoma propagation and monoclonal antibody production.

A major variable to consider in the production of biologicals from mammalian cell cultures is the mode of operation, be it a batch, continuous, perfusion, fed-batch or other production method. The final choice must consider a number of fundamental and economic issues. Here we present some antibody production data from different cell lines using different modes of production and discuss the important factors for consideration in choosing a production strategy. It was found that the productivity of batch cultures was lower than that obtained in continuous and perfused cultures, but that productivity could be improved by implementing suitable feeding strategies. The antibody productivity of one cell line, MCL1, during exponential phase was not affected by media type or glucose level. The maximum productivity of two cell lines in continuous culture was found to occur at dilution rates below the maximum, from 0.019 to 0.030 hr-1.

Agitation and aeration effects in suspension mammalian cell cultures.

Three different hybridoma cell lines, grown in serum-free media with different levels of Pluronic F-68, were subjected to a shear force of 0.6 N m(-2). Some protective effect due to the polymer was found, indicating it to be a potentially useful adjuvant in serum-free media. Other observations of liquid and gas effects at the reactor level have been included here. A discussion of the difference between suspension and microcarrier cultures, in relation to hydrodynamic effects, is included.

Quantitative pulsed Doppler measurement of common femoral artery blood flow variables during postocclusive reactive hyperemia.

Quantitative blood flow measurements were performed on 37 normal lower limbs with a 128-channel digital pulsed Doppler (MDPD) system. The evolution of mean flow (QM), peak systolic flow (QS), diastolic flow (QD), and prograde stroke volume (PSV) was observed at rest, during postocclusive reactive hyperemia (PORH), and at 1-, 2-, and 3-min intervals. The QM at rest was 2.9 +/- 1.1 ml/s; PORH induced a four- to fivefold increase in QM and PSV secondary to a slight increase in QS and the disappearance of the reverse protodiastolic component of resting flow. Reverse flow was restored after 1 min. Both QS and QD returned to resting values after 2 min, whereas QM remained significantly higher after 3 min. To provide a better description of the hyperemic response, we also studied the evolution of the pulsatility index as as applied the flow curve (PIQ). Similarly, the systolic amplitude index (SAI) is presented. Our study demonstrates that pulsed Doppler techniques can be used for noninvasive quantitative assessment of blood flow at rest and during PORH. The values obtained on normal subjects provide base-line data for further investigation of pathological conditions.

[Quantitative measurement of blood flow with an 128-channel Doppler device. Present status of research and future outlook]. / Quantitative Messung der Blutströmung mit einem 128-kanaligen Doppler-Gerät. Derzeitiger Forschungsstand und Zukunftsaussichten.

The recent development of a new multigate pulsed Doppler system used in conjunction with an A-mode scan allows real time display of the velocity profiles across the vessel and quantitative flow measurement. Experimental in-vitro and in-vivo studies showed an excellent correlation between flow measurements obtained by this noninvasive method and by direct timed collection. Preliminary results of the post-occlusive hyperaemic response in normals and in patients with iliac stenosis are presented. Although no statistical comparison is allowed, it appears that the hyperaemic response is diminished when an iliac stenosis is present. A non-invasive method of quantifying the haemodynamic significance of profunda femoris artery stenosis is described. Finally, the velocity profiles and the flow curves in PTFE grafts were studied and compared to the flow patterns of the normal superficial femoral artery. The differences observed between the two conditions might explain the low patency rate of the synthetic grafts. Other fields of application of the method are suggested. The future development of a Duplex scanner combining B-mode imaging and the multigate Doppler system will allow the exploration of vessels within the abdomen and thorax: portal vein, in situ or transplanted renal arteries, ascending and abdominal aorta.