bluuebunny/crossref_metadata_2025 huggingface dataset:
Carbohydrate microarrays and the unravelling of ligands for effector proteins of the immune system
Authors: Feizi Ten
Published: August 2004

Optimism (Feizi 1985) that the carbohydrate sequences of glycoproteins, glycolipids and proteoglycans are bearers of crucial biological information is being well borne out (Lindahl et al. 1994; Feizi 2000; Crocker 2001). Knowledge that the human genome encodes no more than 30 000–50 000 proteins has, in the mean time, served to emphasize the importance of oligosaccharide chains as modulators of the activities and functions of the proteins in health and disease, through recognition processes mediated by carbohydrate‐recognizing receptors. The challenge is to develop high throughput technologies to determine the roles of specific oligosaccharide sequences as ligands for carbohydrate‐recognizing proteins. The neoglycolipid (NGL) technology (Feizi et al. 1994) for generating lipid‐linked oligosaccharide probes has many features that render it adaptable for this challenge. This technology is now the basis of a carbohydrate array system that is applicable both to structurally defined oligosaccharides and to those derived from biological sources, glycoproteins, proteoglycans, cells and even whole organs (Fukui et al. 2002). We observe that such repertoires of NGLs are robust probes when presented on nitrocellulose membranes, thus permitting sensitive and high throughput detection of ligands for carbohydrate‐binding proteins. We show that carbohydrate‐recognizing proteins single‐out their ligands, not only in the arrays of homogenous oligosaccharides, but also in arrays of heterogeneous oligosaccharides. The unique feature is that deconvolution strategies are included with thin‐layer chromatography (TLC) and mass spectrometry for sequencing the ligand‐positive components. I shall discuss the applications in elucidating structures of carbohydrate differentiation antigens and the ligands of carbohydrate‐binding receptors of the immune system.
Changes in mucin‐type O‐glycosylation in breast cancer: implications for the host immune response
Authors: Taylor‐Papadimitriou Joyce , Burchell Joy M. , Miles David , Sewell Robert
Published: August 2004

The major epithelial mucin, which is expressed by more than 90% of breast carcinomas and epithelial breast cancer cell lines, is MUC1. The MUC1 membrane mucin has a relatively simple structure, where a tandem repeat (TR) region carrying multiple O‐glycans forms the major part of the extracellular domain. The gene shows a size polymorphism, due to variation in the number of tandem repeats, each of which has five potential glycosylation sites. MUC1 has been found to be a good model for studying mucin‐type O‐glycosylation both in vitro and in vivo.

Changes in the pattern of glycosylation of MUC1 have been observed in breast cancer (Burchell et al. 2001) and have led to an interest in MUC1 as a possible target antigen for immunotherapeutic intervention (Taylor‐Papadimitriou et al. 2000). The mucin expressed at high levels in the normal lactating mammary gland carries mainly core 2‐based O‐glycans (2–3 per tandem repeat). In breast cancers, the level of the ST3 Gal 1 enzyme is elevated (Burchell et al. 1999) and this is associated with increased numbers of Sialyl T O‐glycans being added, as predicted from in vitro studies, showing competition for the core 1 substrate between this sialyl transferase and the relevant core 2 enzyme (Dalziel et al. 2001). Moreover, the TR domain is more densely glycosylated. While the Sialyl T O‐glycan is commonly found on normal cells (e.g. resting T cells), it is cancer associated in the mammary gland. However, several lines of evidence indicate that this MUC1 glycoform, as expressed on breast cancers, can be immunosuppressive, maybe reflecting an inhibitory interaction with a lectin, possibly a siglec (Nath et al. 1999), on immune effector cells. In 25–30% of breast cancers, O‐glycosylation is terminated very early so that GalNAc (Tn) or NeuAc α2,6 GalNAc (Sialyl Tn) glycans appear. Sialyl Tn is highly tumour‐specific and is detected immunohistochemically only when the enzyme ST6GalNAcI is expressed. The Tn and Sialyl Tn will bind to different lectins, including MGL and could stimulate the immune response. Preliminary studies in mouse models suggest that an immunization protocol which includes MUC1‐carrying Sialyl Tn O‐glycans can protect against tumour growth, while clinical studies in breast cancer patients using immunogens based on the Sialyl Tn glycan are underway.
Molecular recognition and modulation of hepatocyte growth factor activity by heparan and dermatan sulfates
Authors: Catlow Krista , Deakin Jon A. , Delehedde Maryse , Fernig David G. , Pavao Mauro S.G. , Gallagher John T. , Lyon Malcolm
Published: August 2004

Introduction  Hepatocyte growth factor/scatter factor (HGF/SF) is an unusual growth factor in that it binds both heparan sulfate (HS) (Lyon et al. 1994) and dermatan sulfate (DS) (Lyon et al. 1998) glycosaminoglycans (GAGs) with similar high affinities. Both these GAGs act as co‐receptors for HGF/SF in the activation of the Met receptor (Lyon et al. 2002). Our aim was to determine the sequences in HS and DS that specifically interact with and modulate HGF/SF activity.

Materials and methods  A structurally unique DS, which possesses O‐sulfation at carbon‐6 of the hexosamine residue (and not carbon‐4 as in mammalian DS), was obtained from the sea cucumber, Ascidia nigra. A variety of HS‐ and DS‐like structures were also generated using various chemical modification procedures (specific desulfations and carboxyl reductions). The ability of these various GAG species to compete with cell surface GAGs for HGF/SF binding was tested using radiolabelled HGF/SF and MDCK cells. The modified GAG structures and the A. nigra DS are currently being tested for their ability to act as co‐receptors for the interaction between HGF/SF and Met by studying cell signalling and cellular response assays, using the sulfated GAG‐deficient CHO‐745 cell line.

Results  Unexpectedly, A. nigra DS was found to bind HGF/SF strongly with a KD of around 1 nm. This interaction is 20‐fold stronger than that of between HGF/SF and mammalian DS, but similar to that of with HS. A. nigra DS also stimulated HGF/SF‐mediated Erk activation and migration in CHO‐745 cells. Studies using the modified GAG species showed that, in the case of HS, 6‐O‐sulfate and N‐sulfate groups are most important for HGF/SF binding. For HGF/SF binding to DS, hexosamine O‐sulfate is most important. HGF/SF was also found to bind 6‐O‐sulfated GAGs more strongly than 4‐O‐sulfated ones.

Discussion  The data show that there is flexibility in the structures recognized by HGF/SF, and this explains the ability of the growth factor to bind both HS and DS. However, there are still observable preferences in GAG structure, such as 6‐O‐sulfation over 4‐O‐sulfation. Information on HGF/SF‐binding GAG structures is valuable for the design of HGF/SF antagonists that could be useful therapeutically in the treatment of solid tumours where HGF/SF‐Met activity is up‐regulated.
Oligomeric collagen XVIII‐derived endostatin requires cell surface heparan sulfate in order to induce morphological changes in endothelial and epithelial cell lines in vitro
Authors: Clamp A.R. , Blackhall F.H. , Henrioud A. , Jayson G.C. , Javaherian K. , Esko J. , Gallagher J.T. , Merry C.L.R
Published: August 2004

Introduction  Monomeric endostatin (HEM) has been extensively investigated as an endogenous anti‐angiogenic molecule. In contrast, oligomeric endostatin (HED) induces a promigratory phenotype in endothelial cells in vitro that can be inhibited by an EM in a putative negative feedback loop (Kuo et al. 2001). Endostatin is heparin‐binding but the role of cell‐surface glycosaminoglycans (GAG) in regulating the activity of HEM/HED is unclear.

Methods and results  Bovine aortic endothelial (BAE) cells plated onto a Matrigel substratum spontaneously form capillary‐like tubules. Addition of 50 nm HED, 16 h after plating induces the migration of BAE cells away from these tubules. This pro‐migratory action can be inhibited by coincubation with HEM. We have demonstrated that exogenous GAGs can also inhibit the actions of HED. This property is size dependent, with heparin‐derived oligosaccharides containing more than 20 monosaccharide units being optimal inhibitors. HED (50 nm) was also shown to induce marked morphological changes in the Chinese hamster ovary (CHO) epithelial cell line plated in Matrigel, i.e. cell elongation, lamellipodia formation and intercellular bridging. This novel observation allowed us to address the role of cell surface GAGs in HED function using a panel of CHO mutants with defined defects in GAG biosynthesis. CHO 745 cells, which lack all cell surface sulfated GAGs, fail to respond to HED stimulation, while CHO pgsD cells, which specifically lack cell surface heparan sulfate (HS), displayed an attenuated phenotype on HED stimulation with less cell elongation/intercellular bridging. Notably, CHO pgsF17 cells, which lack 2‐O‐sulfation of HS, respond normally to HED indicating that HS 2‐O‐sulfate groups are not necessary for the bioactivity of HED.

To investigate the endostatin–GAG interaction further, zero‐length cross‐linking was utilized. This indicated that multiple HEM subunits can simultaneously bind to a single oligosaccharide chain with up to 3 HEM molecules cross‐linking to a heparin dp22. HED cross‐linking experiments indicate that both endostatin subunits in this molecule may bind to the same oligosaccharide chain. These results suggest that one potential anti‐angiogenic mechanism of HEM is to block the GAG‐binding sites for HED.

Discussion  Cell surface GAGs are absolutely necessary for the bioactivity of HED. HS is required for a wild‐type response, although 2‐O‐sulfation is not essential. The ability to use a panel of GAG‐mutant CHO cells provides a well‐understood system in which to investigate the role of HS in HEM : HED regulation.
Role of heparan sulfate in embryoid body formation
Authors: Drummond K.J , Yates E.A. , Murray P.A. , Turnbull J.E.
Published: August 2004

Introduction  Embryoid bodies (Ebs) are layered, ordered aggregates of cells which form when murine embryonic stem (ES) cells are grown in suspension. Ebs are made up of primitive endoderm cells overlaying an epithelium of epiblast cells, separated by a basement membrane and surrounding a central cavity. The generation of Ebs from undifferentiated ES cells has been used as a model for early development. The process of EB formation has been shown to require signalling through fibroblast growth factor (FGF) receptor 2, implicating heparan sulfate (HS) which is a necessary coreceptor for the formation of a signalling complex.

Heparan sulfate proteoglycans are found both on the surface of cells and in the extracellular matrix. Within the cells, a newly synthesized HS chain is partially modified by a family of enzymes, many of which have multiple isoforms with differing substrate preferences. These modifications include the addition of sulfate groups at up to four positions on each disaccharide unit. The pattern of modifications that a HS saccharide undergoes alters its structure, affecting the ability of the HS to bind and regulate different FGF‐FGF receptor combinations, and so regulate signalling outcomes.

It is hypothesized that the complement of modification enzymes that a cell expresses determines the structure of the HS that the cell produces, thereby altering the signalling response of the cell to HS‐binding factors such as FGFs. In this way, an ES cell could alter its HS to allow signalling by, for example, a particular FGF, setting in motion a chain of intracellular events leading to differentiation.

Materials and methods  Methods used in this study include tissue culture, RT‐PCR and immunocytochemistry.

Results  We have found that undifferentiated ES cells express HS as observed by cell staining with anti‐HS antibodies. They also display particular expression patterns of the different HS modification enzyme isoforms as measured by RT‐PCR. Studies on alterations in HS synthesis and structure during the process of EB formation are underway. In addition, the effect of the addition of exogenous heparin (a highly sulfated analogue of HS) and HS saccharides with variant structures on EB formation is being investigated.

Discussion  Alterations in the profile of modification enzymes expressed by ES cells as they differentiate may correlate with changes in the structure of the HS they make. These changes may allow the differentiating cells to control the way in which they respond to their environment.

These studies will allow the dissection of the structural requirements for a functional role of HS in embryoid body formation and stem cell differentiation.
The use of SA‐β‐Gal to assess cell senescence in intervertebral disc cells
Authors: Evans E.H. , Kletsas D. , Urban J. , Menage J. , Roberts S.
Published: August 2004

Introduction  The intervertebral disc is reported to age faster than other connective tissues with significant degenerative changes already seen in the second decade of life. In other tissues, senescent cells have an altered phenotype, often with a decreased synthetic ability and response to anabolic cytokines. They appear to contribute to age‐related pathologies such as the degeneration of articular cartilage in osteoarthritis. Little is known of cell senescence in the intervertebral disc. In this study, we have investigated the production of senescence‐associated‐ß‐galactosidase (SA‐ß‐Gal), a ‘biomarker’ of cell senescence, in intervertebral disc cells both in cultured populations in vitro, and in vivo, in pathological human discs.

Material and methods  Intervertebral disc cells were extracted from coccygeal bovine discs and cultured for 3 months. Confluent cell preparations were stained for SA‐β‐Gal (Fenton et al. 2001) at passages 0, 1, 2, 3 and 4. Preparations were fixed in 3% paraformaldehyde in phosphate‐buffered saline (PBS) for 5 min at room temperature and incubated for 24 h at 37 °C in SA‐ß‐Gal solution containing 1 mg/ml 5‐bromo‐4‐chloro‐3‐indolyl ß‐d‐galactopyranoside (X‐Gal, Sigma, Poole, UK), 5 mmol/l potassium ferrocyanide, 5 mmol/l potassium ferricyanide, 150 mmol/l NaCl, 2 mmol/l MgCl2 and 40 mmol/l trisodium citrate, titrated with NaH2PO4 to pH 6.0. Lysosomal (nonsenescent) ß‐galactosidase activity was detected using the same solution but titrated to pH 4.0. After staining, preparations were rinsed in ice‐cold PBS, dehydrated and mounted. Pathological human disc from patients with disc herniations or discogenic back pain were also stained for SA‐β‐Gal (immersing the tissue in the stain solution overnight) then cryosectioning (10 µm thick) and fixing. Articular cartilage was studied for comparison (Price et al. 2002).

Results  Cultured intervertebral discs demonstrated some staining for SA‐ß‐Gal at all passages investigated, but there was little change in staining with passage number. Cells in most pathological human discs demonstrated staining for SA‐ß‐Gal. Positive cells were seen more commonly in herniated discs [7/9 (78%); mean age: 46 ± 13] than in those removed from patients with discogenic back pain [2/6 (33%); mean age: 32 ± 5].

Discussion  To our knowledge, this is the first study of cell senescence in intervertebral disc cells. The greatest level was seen in tissue from herniated discs where cell cluster formation and cell proliferation are common (but the mean age of the herniation group was slightly higher than the discogenic back pain group). However, for a tissue demonstrating such significant age‐related degenerative changes, there is surprisingly little expression of SA‐ß‐Gal in comparison with that found in other pathological cartilages. These preliminary data suggest that, unlike the situation in osteoarthritis, early cell senescence is not a major contributing factor in the pathogenesis of disc degeneration.
The effects of human intervertebral disc aggrecan on neuronal and endothelial cell growth
Authors: Johnson W.E.B. , Caterson B. , Eisenstein S.M. , Snow D.M. , Roberts S.
Published: August 2004

Introduction  The development of ‘discogenic’ low back pain has been associated with increased nerve growth into degenerated intervertebral discs, particularly in neovascularized areas. A marked loss of proteoglycans, particularly from the disc's inner regions, is also a marked feature of the degenerative process. As proteoglycans from other tissues can have inhibitory effects on nerve growth, we have hypothesized that the alterations in proteoglycan content seen in disc degeneration may affect the growth of nerves and blood vessels into the disc. To test our hypothesis, we have established cell culture assays to determine the effects of human intervertebral disc aggrecan, which forms the major proteoglyan found in the disc, on neuronal and endothelial cell growth.

Materials and methods  Aggrecan (A1D1 preparations) isolated from the outer (anulus fibrosus, AF) and inner (nucleus pulposus, NP) regions of human lumbar intervertebral discs was incorporated into culture substrata, using methods previously described (Snow et al. 1990). Chick dorsal root ganglia (DRG) and the cell line SH‐SY5Y were used as models of nerve growth. HMEC‐1 and Eahy‐926 cell lines were used as models of endothelial cell growth.

Results  Human disc aggrecan inhibited SHSY‐5Y cell attachment, SHSY5Y neurite outgrowth and induced sensory DRG neurite growth cone turning in a concentration‐dependent manner. Sensory neurites emanating from DRG across permissive substrates (collagen/laminin) were repelled by high (1 mg/ml), but not low (0.01 mg/ml) concentrations of disc aggrecan and became aligned to the aggrecan border. Disc aggrecan similarly inhibited endothelial cell adhesion, cell spreading and migration. HMEC‐1 and Eahy926 cells migrated over collagen substrates (type I) until they encountered disc aggrecan, where they either stopped migrating or, more commonly, changed their direction of movement and aligned to the aggrecan border. In general, aggrecan isolated from the AF was more inhibitory than that isolated from the NP. The inhibitory effects were partially abrogated following enzymic deglycosylation of the aggrecan, or if aggrecan was added in solution (i.e. was nonsubstrate bound).

Conclusions  This study provides evidence that disc aggrecan inhibits neuronal and endothelial growth and migration and therefore supports a hypothesis that a loss of aggrecan from degenerated discs predispose the tissue to vascular and neuronal invasion.
On the Use of Spline Smoothing in Estimating Hedonic Housing Price Models: Empirical Evidence Using Hong Kong Data
Authors: Bao Helen X.H. , Wan Alan T.K.
Published: August 2004

Traditionally in estimating hedonic housing price functions, investigators use parametric models involving specific functional forms and a finite number of unknown parameters. Some investigators have questioned whether the underlying theory is capable of conveying sufficient information to enable a correct and successful specification of parametric models and have instead proposed the less restrictive semiparametric approach to the problem. In this paper, we illustrate how the technique of smoothing splines can be used to estimate hedonic housing price models. Smoothing splines are a powerful approach to the analysis of housing data as they are exceptionally flexible in their functional forms and provide a computationally tractable method even with a large number of explanatory variables. Our illustration takes the form of a rather limited, but very promising, application with Hong Kong data. In the forecasting comparison, the spline smoothing procedure outperforms the traditional parametric Box–Cox model in mean square error terms for out‐of‐sample predictions. Our results also suggest that the distortion caused by underfitting the model is smaller for spline smoothing than for the kernel and k‐nearest‐neighbor semiparametric procedures.
Intra‐abdominal hypertension and the abdominal compartment syndrome
Authors: Hunter J. D. , Damani Z.
Published: August 2004

The pressure within the abdominal cavity is normally little more than atmospheric pressure. However, even small increases in intra‐abdominal pressure can have adverse effects on renal function, cardiac output, hepatic blood flow, respiratory mechanics, splanchnic perfusion and intracranial pressure. Although intra‐abdominal pressure can be measured directly, this is invasive and bedside measurement of intra‐abdominal pressure is usually achieved via the urinary bladder. This cheap, easy approach has been shown to produce results that correlate closely with directly measured abdominal pressures. Significant increases in intra‐abdominal pressure are seen in a wide variety of conditions commonly encountered in the intensive care unit, such as ruptured aortic aneurysm, abdominal trauma and acute pancreatitis. Abdominal compartment syndrome describes the combination of increased intra‐abdominal pressure and end‐organ dysfunction. This syndrome has a high mortality, most deaths resulting from sepsis and multi‐organ failure. Detection of abdominal compartment syndrome requires close surveillance of intra‐abdominal pressure in patients thought to be at risk of developing intra‐abdominal hypertension. The only available treatment for established abdominal compartment syndrome is decompressive laparotomy. Prevention of abdominal compartment syndrome after laparotomy by adoption of an open abdomen approach may be preferable in the patient at significant risk of developing intra‐abdominal hypertension, but this has not been demonstrated in any large trials. Most surgeons prefer to adopt a ‘wait and see’ policy, only intervening when clinical deterioration is associated with a significant increase in intra‐abdominal pressure.
Antibiotic‐treated infections in intensive care patients in the UK
Authors: Cuthbertson B. H. , Thompson M. , Sherry A. , Wright M. M. , Bellingan G. J. ,   the Intensive Care Society
Published: August 2004

The purpose of this audit was to study reasons for starting antibiotic therapy, duration of antibiotic treatment, reasons for changing antibiotics and the agreement between clinical suspicion and microbiological results in intensive care practice. We conducted a multicentre observational audit of 316 patients. Data on demographic details, site, treatment and nature of infection were collected. The median duration of antibiotic therapy was 7 days. Infections were community‐acquired in 160 patients (55%). Antibiotics were started on clinical suspicion of infection in 237 patients (75%). Pulmonary infections were the most common, representing 52% of all proven infections. Gram‐negative organisms were the most common cause of proven infections (n = 90 (50%)). The antibiotic spectrum was narrowed in light of microbiology results in 78 patients (43%) and changed due to antibiotic resistance in 38 patients (21%). We conclude that the mean duration of treatment contrasts with existing published guidelines, highlighting the need for further studies on duration and efficacy of treatment in intensive care.
Intracranial pressure measurement, induced hypothermia and barbiturate coma in meningitis associated with intractable raised intracranial pressure
Authors: Cuthbertson B. H. , Dickson R. , Mackenzie A.
Published: August 2004

We report the use of intracranial pressure monitoring, mild hypothermia and barbiturate coma in a patient with meningococcal meningitis complicated by raised intracranial pressure.
A partially blinded randomised controlled trial of patient‐maintained propofol sedation and operator controlled midazolam sedation in third molar extractions
Authors: Leitch J. A. , Anderson K. , Gambhir S. , Millar K. , Robb N. D. , McHugh S. , Kenny G. N. C.
Published: August 2004

Patient‐maintained sedation using propofol has recently been shown to be effective for dental surgery. We compared this new technique to the established technique of operator administered midazolam. The two groups were compared before, during and after sedation. The two primary outcomes were time until discharge and oxygen saturation. Vital signs, anxiety and psychomotor skills were also compared. State anxiety was reduced to a greater extent in the propofol group (mean difference 10 (SD 4) mm; p = 0.010. Propofol patients recovered quicker (mean difference 7 (SD 1.4) min; p = 0.001). Propofol patients had a smaller reduction in arterial oxygen saturation (mean difference 0.8 (SD 0.3)%; p = 0.030), and a reduced increase in heart rate (mean difference 9 (SD 2) beats.min–1; p < 0.001). Both techniques were well tolerated and safe. Propofol sedation offered superior anxiolysis, quicker recovery, less amnesia and less depression of simple psychomotor function.
The effect of a mechanical glottis on peak expiratory flow rate and time to peak flow during a peak expiratory flow manoeuvre: A study in normal subjects and patients with motor neurone disease
Authors: Suleman M. , Abaza K. T. , Gornall C. , Kinnear W. J. M. , Wills J. S. , Mahajan R. P.
Published: August 2004

We have evaluated a mechanical glottis in healthy volunteers and in patients with bulbar motor neurone disease. In healthy volunteers, the mechanical glottis increased peak flow rate and decreased the time to peak flow during forced expiration, but cough produced even higher flow rates and shorter times to peak flow. In patients, the mechanical glottis increased peak flow rate and decreased the time to peak flow. The mechanical glottis also produced higher peak flow rates when compared to the cough manoeuvres, and the time to peak flow was also significantly shorter with the mechanical glottis. We have shown that the use of a mechanical glottis tends to convert the airflow profile of a peak expiratory flow manoeuvre into that of a cough in both healthy volunteers and patients with motor neurone disease. Its potential role as an aid to clearance of airway secretions in patients with impaired laryngeal function remains to be seen.
The effect of celecoxib on intrathecal morphine‐induced pruritus in patients undergoing Caesarean section
Authors: Lee L. H. Y. , Irwin M. G. , Lim J. , Wong C. K.
Published: August 2004

Pruritus associated with intrathecal opioid administration is particularly common in pregnancy. Nonsteroidal anti‐inflammatory drugs (NSAIDs) can reduce the severity of this pruritus but have undesirable side effects. The recent development of drugs that can specifically inhibit the cyclooxygenase 2 isoenzyme have become an attractive alternative. This study was designed to evaluate the efficacy of such a drug (celecoxib) in reducing intrathecal opioid‐induced pruritus in a randomised double‐blinded study of 60 women undergoing Caesarean section. All of them received spinal anaesthesia with 3 ml of 0.5% hyperbaric bupivacaine and 0.3 mg preservative‐free morphine. After delivery of the baby, they received either oral celecoxib 200 mg or placebo. Visual analogue scores for pain and pruritus were measured at 30 min, 2, 4, 8, and 24 h. There was no difference in the severity and onset of pain and pruritus between the two groups. Timing of administration, inadequate dosing and possible altered pharmacokinetics in pregnancy may explain the lack of efficacy.
Fluxes of N2O from farmed peat soils in Finland
Authors: Regina K. , Syväsalo E. , Hannukkala A. , Esala M.
Published: August 2004

Agricultural peat soils are important sources of nitrous oxide (N2O). Emissions of N2O were measured from field plots of grass, barley, potatoes and fallow on a peat field in northern Finland during 2000–2002 and in southern Finland in 1999–2002. In the north the mean annual fluxes of N2O (with their standard errors) during 2 years were 4.0 (±1.2), 13 (±3.0) and 4.4 (±0.8) kg N ha−1 from the plots of grass, barley and fallow, respectively. In the north there were no significant thaw periods in the middle of winter. As a result, the thawing in the spring did not induce especially large N2O emissions. Emissions of N2O were larger in the south than in the north. In the southern peat field the mean annual fluxes during 3 years were 7.3 (±1.2), 15 (±2.6), 10 (±1.9) and 25 (±6.9) kg N2O‐N ha−1 for grass, barley, potato and fallow plots, respectively. Here, the largest single episodes of emission occurred during the spring thaw each year, following winter thaw events. An emission factor of 10.4 kg N2O‐N ha−1 year−1 for the N2O emission from the decomposition of the peat results from these data if the effect of fertilization according to the IPCC default emission factor is omitted. The direct effect of adding N as fertilizer on N2O emissions was of minor importance. On average, 52% of the annual N2O flux entered the atmosphere outside the cropping season (October–April) in the north and 55% in the south. The larger N2O fluxes from the peat soil in the south might be due to the more humified status of the peat, more rapid mineralization and weather with more cycles of freezing and thawing in the winter.
Thymocytes induced by antigen injection into the anterior chamber activate splenic CD8+ suppressor cells and enhance the antigen‐induced production of immunoglobulin G1 antibodies
Authors: Li Xingya , Wang Yafei , Urso David , O'Rourke James , Cone Robert E.
Published: August 2004

Injection of antigen into the ocular anterior chamber (AC) of a mouse eye (an immunologically privileged site) induces the activation of immunoregulatory NK1.1+, CD4– CD8–, T‐cell receptor (TCR) αβ+ thymocytes. These thymocytes transfer the suppression of delayed‐type hypersensitivity (DTH) when injected into mice sensitized to the same antigen but do not effect the suppression of DTH. On the other hand, the immunized recipients of these transferred thymocytes produce splenic CD8+ T cells that effect the suppression of DTH. However, it is unclear whether the thymocytes transferred from the AC‐injected donor differentiate into and/or activate CD8+ T‐splenic suppressor cells. We therefore sought to determine the origin of splenic suppressor cells produced in the recipients of immunoregulatory thymocytes transferred from donors that receive an injection of antigen into the AC. CD45.1+ thymocytes from mice that received an AC injection of 2,4,6‐trinitrobenzene sulphonic acid (TNP)‐bovine serum albumin (BSA) were transferred to congenic CD45.2+ TNP‐BSA‐immune recipients. Spleen cells from the recipients were then sorted based on anti‐CD45.1 or ‐CD45.2 antibody binding and assayed for suppressor cells. This was done by the injection of separated spleen cells into the footpad of TNP‐BSA‐immunized mice, concurrent with the induction of footpad swelling (contact sensitivity) of the footpad elicited by an epicutaneous application of picryl chloride. The systemic distribution of antigen after the injection of antigen into the AC was demonstrated by the injection of fluorescein or 125I‐labelled TNP‐BSA into the AC. The results demonstrate that (i) splenic CD8+ T‐suppressor cells produced in the immunized recipients of immunoregulatory thymocytes are derived from the CD45.2 recipient of the CD45.1+ thymocytes; (ii) the induction of recipient splenic suppressor T cells by the transferred immunoregulatory thymocytes requires that the recipient be immunized to the same antigen as that used to induce immunoregulatory thymocytes; (iii) antigen is introduced to the thymus after an injection of antigen into the AC; (iv) although the transfer of the suppression of DTH by regulatory thymocytes was not dependent on interleukin‐4 (IL‐4), CD4+ NK1.1– regulatory thymocytes from AC‐injected donors enhanced the production of immunoglobulin G1 antibodies to TNP‐BSA by an IL‐4‐dependent mechanism. These observations suggest that the adult thymus plays an active role in the induction and maintenance of anterior chamber‐associated immune deviation as manifested by the generation of the suppression of cell‐mediated immunity to exogenous antigen and the antigen‐induced production of IgG1 antibodies.
Immunosuppressive therapy modulates T lymphocyte gene expression in patients with systemic lupus erythematosus
Authors: Pereira Elayne , Tamia‐Ferreira Márcia Cristina , Cardoso Renato Sousa , Mello Stephano Spanó , Sakamoto‐Hojo Elza Tiemi , Passos Geraldo A. S. , Donadi Eduardo Antonio
Published: August 2004

To evaluate the T‐cell large‐scale differential gene expression in systemic lupus erythematosus (SLE) patients presenting with glomerulonephritis we studied SLE patients before and after immunosuppressive treatment. Large‐scale gene expression of peripheral blood mononuclear T cells was evaluated using cDNA microarray nylon membranes containing 5184 cDNAs. Data were analysed using the SAM and Cluster and Treeview software. When untreated patients were compared to healthy individuals, 38 genes, most of them located on chromosomes 1, 3, 6, 17 and 19, were repressed, and when untreated patients were compared to treated ones, 154 genes, located on chromosomes 1, 6, 7, 12 and 17, were induced. In terms of biological function of coded proteins, the differentially expressed genes were associated with apoptosis, cell cycle, chromosomal scaffold, cytokine/chemokine, DNA repair/replication, Golgi/mitochondrial proteins, mRNA processing, signalling molecules and tumour suppressors. Two autoantigen genes related to RNA splicing (small nuclear riboprotein 70 000 MW‐U1 SNR, and splicing factor 3a, 60 000 MW), and the tetranectin‐plasminogen‐binding protein were repressed. The Fc fragment of immunoglobulin G low affinity IIb, apoptotic protease activating factor‐1, two subunits of cytochrome c, caspase 8, complement C5a, HLA‐DRA, HLA‐DQB1, transforming growth factor‐β receptor II, small nuclear ribonucleoprotein polypeptide N (Sm protein N) genes, heterogeneous nuclear riboprotein‐C, and argininosuccinate lyase genes, among others, were induced. A total of 10 genes were repressed in untreated patients and induced in treated ones, among them tumour necrosis factor (ligand) superfamily member 9, tumour protein p53, mannosidase alpha class IA, and CD22. Although some of these differentially expressed genes are typically expressed in B cells, CD22 and CD32 have also been reported in T cells and may provide regulatory signals to B cells. Assessment of differential gene expression may provide hybridization signatures that may identify susceptibility, diagnostic and prognostic markers of SLE.
TLR9−/− and TLR9+/+ mice display similar immune responses to a DNA vaccine
Authors: Babiuk Shawn , Mookherjee Neeloffer , Pontarollo Reno , Griebel Phillip , Van Drunen Littel‐Van Den Hurk Sylvia , Hecker Rolf , Babiuk Lorne
Published: August 2004

Plasmid DNA continues to attract interest as a potential vaccine‐delivery vehicle. However, the mechanisms whereby immune responses are elicited by plasmids are not fully understood. Although there have been suggestions regarding the importance of CpG motifs in plasmid immunogenicity, the molecular mechanisms by which CpG motifs enhance immune responses to DNA vaccines are not well understood. As Toll‐like receptor 9‐deficient (TLR9−/−) mice fail to respond to the adjuvant effects of CpG oligonucleotides, we used these mice to determine the effect of CpG motifs in plasmids used for DNA immunization. In the study described below, we report that DNA immunization was as effective in eliciting antigen‐specific antibody and at stimulating antigen‐specific interferon‐γ (IFN‐γ)‐secreting cells in TLR9−/− mice as in TLR9+/+ mice. This study illustrates that DNA vaccines elicit immune responses by multiple mechanisms and demonstrates that TLR9 is not essential for the induction of immune responses following DNA immunization.
Asymptomatic parasitaemia as a risk factor for symptomatic malaria in a cohort of Ugandan children
Authors: Njama‐Meya Denise , Kamya Moses R. , Dorsey Grant
Published: August 2004

Objectives  To assess the prevalence of asymptomatic parasitaemia, determine its association with symptomatic malaria, and identify independent predictors of asymptomatic parasitaemia in a cohort of children from Kampala, Uganda.

Methods  A total of 316 children aged 6 months to 5 years were recruited from the community. The prevalence of asymptomatic parasitaemia was assessed at enrolment and approximately every 30 days during follow‐up. Participants received all of their health care in our clinic, including a standardized approach to the diagnosis and treatment of symptomatic malaria.

Results  A total of 283 (90%) subjects completed the full 1‐year follow‐up and were included in this study, yielding 2557 routine smears. The prevalence of asymptomatic parasitaemia was 17% at enrolment, but 5–8% for the remainder of the study. The risk of developing symptomatic malaria within 30 days was significantly higher in those with a positive routine than in those with a negative one (50%vs. 9%, P < 0.001). Higher parasite densities were associated with increased odds of developing symptomatic malaria within 30 days (P = 0.003). Only 11% of episodes of asymptomatic parasitaemia, involving 6% of subjects, arose and cleared without therapy. In multivariate analysis the only significant risk factor for asymptomatic parasitaemia was whether a child had any episode of symptomatic malaria during the course of the study (OR = 3.0, P = 0.02).

Conclusion  In our cohort of children from an urban meso‐endemic environment, asymptomatic parasitaemia was uncommon and frequently followed by symptomatic malaria. This suggests that presumptive treatment of asymptomatic parasitaemia in such settings would be an efficient means of preventing symptomatic malaria.
Investigational use of ribavirin in the treatment of severe acute respiratory syndrome, Singapore, 2003
Authors: Leong Hoe‐Nam , Ang Brenda , Earnest Arul , Teoh Cindy , Xu Wei , Leo Yee‐Sin
Published: August 2004

Objective  Ribavirin is a broad spectrum nucleoside analogue efficacious in the treatment of several viral infections. In the recent severe acute respiratory syndrome (SARS) outbreak, ribavirin was used in various countries against this novel coronavirus. We conducted a retrospective analysis to assess the efficacy of ribavirin in the treatment of SARS in Singapore.

Methods  A total of 229 cases were analysed. Ninety‐seven (42.4%) patients received ribavirin at a mean of 6.4 days of illness. Clinical and laboratory parameters taken at the start of ribavirin treatment were compared with day 5, 6 or 7 parameters of those patients not on treatment. The two groups were compared using Fisher's exact test and Student's t‐test. Multivariate analysis was performed using Cox regression model with death as the outcome of interest.

Results  The treatment group had younger women with more symptoms of myalgia (P < 0.001). The crude death rate was 12.9% in the control and 10.3% (P = 0.679) in the treatment group. After correction for age, male sex, lactate dehydrogenase levels and steroid use, the hazard ratio was 1.03 (95% CI: 0.44–2.41, P = 0.939).

Conclusion  In this retrospective, uncontrolled cohort analysis, use of ribavirin did not appear to confer any benefit for patients with SARS.
Additional health and development activities for community‐directed distributors of ivermectin: threat or opportunity for onchocerciasis control?
Authors: Okeibunor Joseph C. , Ogungbemi Michael K. , Sama Martin , Gbeleou Sesso C. , Oyene Ukam , Remme Jan H. F.
Published: August 2004

We studied the involvement of community‐directed distributors (CDDs) of ivermectin for onchocerciasis control in other health and development activities in Nigeria, Togo and Cameroon. Most CDDs (82%) were involved in additional activities, especially EPI, water and sanitation and community development projects. These activities did not take up much time and were not considered an important burden. Ivermectin treatment coverage did not decline with increasing number of additional activities. Other health programmes are interested in building on the experiences and structures of community‐directed treatment with ivermectin and involving CDDs in their health programmes. Some, such as EPI, provide financial incentives. Incentives by other programmes may enhance the performance of CDDs when they are selected on the basis of their CDD status. CDDs, health personnel and community members have very positive attitudes towards greater involvement of CDDs in additional health and development activities. We conclude that additional activities for CDDs do not pose a threat to but rather provide an opportunity to strengthen sustainability and effectiveness of ivermectin treatment.
Effective antiplatelet therapy does not prolong transgenic pig to baboon cardiac xenograft survival
Authors: Schirmer Johannes M. , Fass David N. , Byrne Guerard W. , Tazelaar Henry D. , Logan John S. , McGregor Christopher G. A.
Published: August 2004

Abstract:  Background:  Microvascular thrombosis is a prominent characteristic of delayed xenograft rejection, therefore the effects of antiplatelet therapy with aspirin and clopidogrel on long‐term cardiac xenograft function was investigated in a heterotopic pig‐to‐baboon cardiac transplant model.

Methods:  Donor hearts from human CD46 transgenic pigs were transplanted heterotopically to baboons. The recipients received immunosuppression that included tacrolimus, sirolimus, corticosteroids, anti‐CD20 monoclonal antibody and TPC, an α‐galactosyl‐polyethylene glycol conjugate. In group 1 (n = 9) in addition to immunosuppression, the recipients received combination therapy consisting of aspirin (80 mg/day) and clopidogrel (75 mg/day) beginning 2 days after transplant and continuing until cessation of graft function. Antiaggregatory efficacy was evaluated by platelet aggregation assay. In group 2 (n = 9) antiplatelet drugs were not given.

Results:  Functional assays confirmed inhibition of platelet aggregation in group 1 suggesting sufficient systemic effects of the treatment. However, anticoagulant therapy did not result in significant prolongation of xenograft function (group 1: median survival 22 days, range 15 to 30 days; group 2: median survival 15 days, range 4 to 53 days). Histologic analysis at rejection revealed no difference in the level of platelet containing thrombi between the groups.

Conclusions:  Inhibition of platelet aggregation by a combination of aspirin and clopidogrel did not have a significant impact on the length of xenograft survival or on the development of microvascular thrombosis in this pig‐to‐primate model.
Does exposure to swine leukocyte antigens after pig‐to‐nonhuman primate xenotransplantation provoke antibodies that cross‐react with human leukocyte antigens?
Authors: Key Tim , Schuurman Henk‐Jan , Taylor Craig J.
Published: August 2004

Abstract:  Background:  A potential concern of using pig kidney xenografts for human transplantation is that antibodies produced to swine leukocyte antigens (SLA) may cross‐react with human leukocyte antigens (HLA) and thereby limit the scope for a subsequent human organ donor transplant. We therefore investigated whether exposure to SLA after pig‐to‐nonhuman primate kidney xenotransplantation gives rise to HLA cross‐reactive antibodies.

Methods:  Serum samples were obtained from 52 cynomolgus monkeys that received kidney transplants from human decay‐accelerating factor (hDAF) transgenic pigs. Samples were collected pre‐transplant and at time of autopsy (mean 20 days post‐transplantation, range 1 to 53 days) and analyzed for IgG HLA class I and HLA class II specific antibodies by enzyme‐linked immunosorbent assay (ELISA) against pooled purified HLA antigens. To ensure the ability of the HLA ELISA to detect cynomolgus monkey IgG binding, parallel experiments were performed to detect IgG Gal‐α‐1,3‐Gal‐specific antibodies known to be present in cynomolgus monkey serum.

Results:  Analysis of both pre‐ and post‐transplantation serum samples by ELISA demonstrated no detectable IgG antibody binding to HLA class I or class II antigens. Using the same ELISA antibody detection reagents, IgG Gal‐α‐1,3‐Gal‐specific antibodies were identified in 13 of 38 (34%) sera obtained before transplantation and 21 of 52 (40%) sera collected post‐transplantation, confirming that the negativeHLA ELISA results were not due to a technical aspect of the assay.

Conclusion:  This study suggests that exposure to SLA following transplantation of porcine kidneys in nonhuman primates does not give rise to antibodies that cross‐react with HLA.
Loss of complementation and the logic of two-step meiosis
Authors: Archetti M.
Published: August 2004

Meiosis is usually a two-step process: two divisions preceded by a duplication. One-step meiosis, a single division without prior replication, is a more logical way to produce haploid gametes; moreover, one-step meiosis leads to higher variabilty in the progeny than two-step meiosis. Yet one-step meiosis is very rare in nature, and may not even exist at all. I suggest that this is because one-step meiosis, in contrast to two-step meiosis, can be easily invaded and replaced by asexual reproduction. I discuss why other existing peculiar forms of division leading to the production of haploid gametes, but not one-step meiosis, have the same effect as two-step meiosis.
Natural selection, evolvability and bias due to environmental covariance in the field in an annual plant
Authors: Winn A. A.
Published: August 2004

Estimates of the form and magnitude of natural selection based on phenotypic relationships between traits and fitness measures can be biased when environmental factors influence both relative fitness and phenotypic trait values. I quantified genetic variances and covariances, and estimated linear and quadratic selection coefficients, for seven traits of an annual plant grown in the field. For replicates of 50 paternal half-sib families, coefficients of selection were calculated both for individual phenotypic values of the traits and for half-sib family mean values. The potential for evolutionary response was supported by significant heritability and phenotypic directional selection for several traits but contradicted by the absence of significant genetic variation for fitness estimates and evidence of bias in phenotypic selection coefficients due to environmental covariance for at least two of the traits analysed. Only studies of a much wider range of organisms and traits will reveal the frequency and extent of such bias.
Evidence for a single median fin-fold and tail in the Lower Cambrian vertebrate, Haikouichthys ercaicunensis
Authors: Zhang X.-G. , Hou X.-G.
Published: August 2004

In this study, we illustrate an exceptionally well-preserved Haikouichthys ercaicunensis from the Lower Cambrian Chengjiang fauna that displays complete single dorsal, ventral and caudal fins. This 530-million-year old vertebrate is fish-shaped and characterized by a single median fin-fold, which is an essential trait of the initial vertebrate chordates. The radially orientated ray-like structures in its dorsal fin somewhat resemble but are probably not real radials seen in basal vertebrates, such as hagfishes and lampreys. The unique design of primitive fins and fin structures provides additional insights into the early evolution of vertebrates.
Diminishing returns in social evolution: the not-so-tragic commons
Authors: Foster K. R.
Published: August 2004

A challenge for evolutionary theory is to understand how cooperation can occur in the presence of competition and cheating, a problem known as the tragedy of commons. Here I examine how varying the fitness returns from reproductive competition or cooperation affects the negative impact of competition on a social group. Varying linear returns does not affect the impact of competition. However, diminishing returns, where additional investments in either competition or cooperation give smaller and smaller rewards, reduce the effects of competition on the group. I show that diminishing returns are common in many systems, including social vertebrates, microbes, social insects and mutualisms among species. This suggests that the tragedy of the commons is not so tragic and that the disruptive effects of competition upon social life will often be minor.