Tumor associated macrophages (TAM) influence diverse processes such as angiogenesis, tumor cell proliferation, and metastasis during tumor progression. In a variety of tumor types, the amount of TAM has been associated with prognosis, but their role in prostate cancer has not been elucidated. The purpose of this study was to investigate the role of TAM in a series of 85 cases of prostatic carcinoma, diagnosed at transurethral resection of the prostate between 1975-1983, using immunohistochemistry and morphometrical techniques. Macrophage density was assessed as the maximum number of TAM per field in the three most macrophage dense areas (TAMmax) and as the average volume density of TAM in an estimate of the whole resected tumor. Furthermore, the individual cell profile area of TAM was assessed with an image analyzer. Macrophage variables were thereafter related to histological grade, tumor stage, metastasis as well as to vascular density, tumor cell proliferation and survival. Patients with a volume density of TAM in the fourth quartile had a shorter median cancer specific survival time than patients in the first to third quartile (3.3 vs. 5.9 years, p=0.005). Furthermore, an increased macrophage cell profile area was related to poor clinical outcome (4.6 vs. 5.9 years, p=0.039) whereas TAMmax gave no prognostic information. In a multivariate analysis, metastasis and the volume density of macrophages gave independent prognostic information (p=0.0008, p=0.010). However, when excluding metastasis from the analysis, only Gleason score was an independent predictor of cancer specific survival (p= 0.005). The volume density of TAM, the macrophage cell profile area and TAMmax increased with increasing Gleason score (p=0.001, p=0.0001, p=0.0001 respectively). A correlation was found between the volume density of TAM and tumor cell proliferation (rs=0.44, p=0.001) and an increased macrophage cell profile area was associated to microvessel density (rs=0.42, p=0.0001). Together these results suggest that both the functional state (as reflected by cell size), number and location of the macrophages are of importance for their influence on prostate tumors, but macrophage quantification is not a strong independent prognostic factor.

Neuronal apoptosis was observed in the rat dentate gyrus in two experimental models of human limbic epilepsy. Five hours after one hippocampal kindling stimulation, a marked increase of in situ terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) of fragmented DNA was observed in nuclei located within and on the hilar border of the granule cell layer and in the polymorphic region. Forty kindling stimulations with 5-min interval produced higher numbers of labeled nuclei compared with one stimulation. The increase of TUNEL-positive nuclei was prevented by the protein synthesis inhibitor cycloheximide but not affected by the N-methyl-D-aspartate receptor antagonist MK-801. Kainic acid-induced seizures lead to a pattern of labeling in the hippocampal formation identical to that evoked by kindling. A large proportion of cells displaying TUNEL-positive nuclei was double-labeled by the neuron-specific antigen NeuN, demonstrating the neuronal identity of apoptotic cells. Either 1 or 40 kindling stimulations also gave rise to a marked increase of the number of cells double-labeled with the mitotic marker bromodeoxyuridine and NeuN in the subgranular zone and on the hilar border of the dentate granule cell layer. The present data show that single and intermittent, brief seizures induce both apoptotic death and proliferation of dentate gyrus neurons. We hypothesize that these processes, occurring early during epileptogenesis, are primary events in the development of hippocampal pathology in animals and possibly also in patients suffering from temporal lobe epilepsy.

BACKGROUND

Metastatic renal carcinoma has a 2-year survival of around 20% and is largely resistant to chemotherapy. The use of interferons in the treatment of metastatic renal carcinoma remains controversial. Although non-randomised studies suggest that biological therapy with interferons produces a small number of tumour responses, most clinicians judge such treatment to be ineffective. We have investigated the effect of treatment with interferon-alpha on survival in patients with metastatic renal carcinoma.

METHODS

In a multicentre, randomised trial, patients with metastatic renal carcinoma were randomly assigned subcutaneous interferon-alpha (three doses--5 MU, 5 MU, 10 MU--for the first week, then 10 MU three times per week for a further 11 weeks; n=174) or oral medroxyprogesterone acetate (MPA; 300 mg once daily for 12 weeks; n=176). The primary endpoint was overall survival. Analysis was by intention to treat. The trial used a triangular sequential design for early termination as soon as results were conclusive. The trial was stopped in November, 1997, when data were available for 335 patients (167 interferon-alpha, 168 MPA).

RESULTS

A total of 111 patients have died in the interferon-alpha group, and 125 patients have died in the MPA group. There was a 28% reduction in the risk of death in the interferon-alpha group (hazard ratio 0.72 [95% CI 0.55-0.94], p=0.017). Interferon-alpha gave an improvement in 1-year survival of 12% (MPA 31% survival, interferon-alpha 43%), and an improvement in median survival of 2.5 months (MPA 6 months, interferon-alpha 8.5 months).

CONCLUSIONS

The benefit of treatment with interferon-alpha should be weighed against the drug's toxic effects. Combination regimens of biological therapy and chemotherapy should now be compared with interferon-alpha monotherapy in randomised controlled trials.

Mutations in RRP6 result in the accumulation of aberrant polyadenylated transcripts from small nucleolar RNA genes. We exploited this observation to search for novel noncoding RNA genes in the yeast genome. When RNA from rrp6Delta yeast is compared with wild-type on whole-genome microarrays, numerous intergenic loci exhibit an increased mutant/wild type signal ratio. Among these loci, we found one encoding a new C/D box small nucleolar RNA, as well as a surprising number that gave rise to heterogeneous Trf4p-polyadenylated RNAs with lengths of approximately 250-500 nt. This class of RNAs is not easily detected in wild-type cells and appears associated with promoters. Fine mapping of several such transcripts shows they originate near known promoter elements but do not usually extend far enough to act as mRNAs, and may regulate the transcription of downstream mRNAs. Rather than being uninformative transcriptional "noise," we hypothesize that these transcripts reflect important features of RNA polymerase activity at the promoter. This activity is normally undetectable in wild-type cells because the transcripts are somehow distinguished from true mRNAs and are degraded in an Rrp6p-dependent fashion in the nucleus.

alpha 2-Macroglobulin (alpha 2M) was isolated from human plasma by a four-step procedure: poly(ethylene glyco) fractionation, gel chromatography, euglobulin precipitation and immunoadsorption. No contaminants were detected in the final preparations by electrophoresis or immunoprecipitation. The protein ran as a single slow band in gel electrophoresis, and was designated 'S-alpha 2M'. S-alpha 2M bound about 2 mol of trypsin/mol. Treatment of S-alpha 2M with a proteinase or ammonium salts produced a form of the molecule more mobile in electrophoresis, and lacking proteinase-binding activity (F-alpha 2M). The electrophoretic mobility of the F-alpha 2M resulting from reaction with NH4+ salts was identical with that of proteinase complexes. We attribute the change in electrophoretic mobility of the alpha 2M to a conformation change, but there was no evidence of a change in pI or Strokes radius. Electrophoresis of S-alpha 2M in the presence of sodium dodecylsulphate gave results consistent with the view that the alpha 2M molecule is a tetramer of identical subunits, assembled as a non-covalent pair of disulphide-linked dimers. Some of the subunits seemed to be 'nicked' into two-thires-length and one-third-length chains, however. This was not apparent with F-alpha 2M produced by ammonium salts. F-alpha 2M produced by trypsin showed two new bands attributable to cleavage of the subunit polypeptide chain near the middle. Immunoassays of F-alpha 2M gave 'rockets' 12-29% lower than those with S-alpha 2M. The nature of the interactions between subunits in S-alpha 2M and F-alpha 2M was investigated by treating each form with glutaraldehyde before electrophoresis in the presence of sodium dodecyl sulphate. A much greater degree of cross-linking was observed with the F-alpha 2M, indicating that the subunits interact most closely in this form of the molecule. Exposure of S-alpha 2M to 3 M-urea or pH3 resulted in dissociation to the disulphide-bonded half-molecules; these did not show the proteinase-binding activity characteristic of the intact alpha 2M. F-alpha 2M was less easily dissociated than was S-alpha 2M. S-alpha 2M was readily dissociated to the quarter-subunits by mild reduction, with the formation of 3-4 new thiol groups per subunit. Inact reactive alpha 2M could then be regenerated in high yield by reoxidation of the subunits. F-alpha 2M formed by reaction with a proteinase or ammonium salts was not dissociated under the same conditions, although the interchain disulphide bonds were reduced. If the thiol groups of the quarter-subunits of S-alpha 2M were blocked by carboxymethylation, oxidative reassociation did not occur. Nevertheless treatment of these subunits with methylammonium salts or a proteinase caused the reassembly of half-molecules and intact (F-) tetramers. It is emphasized that F-alpha 2M does not have the properties of a denatured form of the protein...

An autoinducer required for the growth-dependent development of luminescence in Vibrio harveyi has been purified, structurally identified, and chemically synthesized. The autoinducer, which is excreted by the cells, was extracted with chloroform from conditioned media in which V. harveyi cells had been grown. The concentrated extract was separated on a silica gel column and the autoinducer activity further purified by thin layer, paper, and high performance liquid chromatography. The structure of the partially purified autoinducer was identified by 1H NMR and mass spectrometry as N-(beta-hydroxybutyryl)homoserine lactone. This compound was chemically synthesized by condensation of beta-hydroxybutyrate with alpha-amino-gamma-butyrolactone hydrobromide using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as a carboxyl group activator. The pure synthetic autoinducer gave the characteristic NMR and mass spectra, co-migrated with the natural autoinducer on thin layer plates, and specifically stimulated induction of luminescence of V. harveyi. Light emission of a regulatory dark mutant of V. harveyi could be stimulated over 1000-fold by the addition of N-(beta-hydroxybutyryl)homoserine lactone, reaching intensities comparable to that of the native strain. The similarity in structure of the autoinducer of V. harveyi to that of Vibrio fischeri suggests that the regulation of luminescence induction in these bacteria may be related in spite of their differences in lux gene organization.

BACKGROUND

Epidemiological studies suggest an association between infection with Epstein-Barr virus (EBV) and risk of multiple sclerosis (MS).

OBJECTIVE

To determine whether elevation in serum antibody titers to EBV viral capsid antigen (VCA), nuclear antigens (EBNA, EBNA-1, and EBNA-2), and diffuse and restricted early antigen (EA-D and EA-R) as well as to cytomegalovirus (CMV) precede the occurrence of MS.

METHODS

Prospective, nested case-control study. Of 62 439 women participating in the Nurses' Health Study (aged 30-55 years in 1976) and Nurses' Health Study II (aged 25-42 years in 1989) who gave blood samples in 1989-1990 and 1996-1999, respectively, and were followed up through 1999, 144 women with definite or probable MS and 288 healthy age-matched controls were included in the analysis.

METHODS

Serum antibody titers to the specific EBV and CMV antigens, compared between cases and controls.

RESULTS

We documented 18 cases of MS with blood collected before disease onset. Compared with their matched controls, these women had higher serum geometric mean titers (GMTs) of antibodies to EBV but not CMV. Elevations were significant for antibodies to EBNA-1 (GMT, 515 vs 203; P =.03), EBNA-2 (GMT, 91 vs 40; P =.01), and EA-D (15.9 vs 5.9; P =.04). The strongest association was found for antibodies to EBNA-2; a 4-fold difference in titers was associated with a relative risk (RR) of MS of 3.9 (95% confidence interval [CI], 1.1-13.7). The corresponding RRs were 1.6 (95% CI, 0.7-3.7) for VCA, 2.5 (95% CI, 1.0-6.3) for EBNA, 1.8 (95% CI, 1.0-3.1) for EA-D, and 1.0 (95% CI, 0.6-1.7) for CMV. Significant but generally weaker elevations in anti-EBV antibodies were also found in analyses of 126 cases of MS with blood collected after disease onset and their matched controls.

CONCLUSIONS

Our results support a role of EBV in the etiology of MS.

Osmotic stress responses are critical not only to the survival of unicellular organisms but also to the normal function of the mammalian kidney. However, the extent to which cells outside the kidney rely on osmotic stress responses in vivo remains unknown. Nuclear factor of activated T cells 5 (NFAT5)/tonicity enhancer binding protein (TonEBP), the only known osmosensitive mammalian transcription factor, is expressed most abundantly in the thymus and is induced upon lymphocyte activation. Here we report that NFAT5/TonEBP is not only essential for normal cell proliferation under hyperosmotic conditions but also necessary for optimal adaptive immunity. Targeted deletion of exons 6 and 7 of the Nfat5 gene, which encode a critical region of the DNA-binding domain, gave rise to a complete loss of function in the homozygous state and a partial loss of function in the heterozygous state. Complete loss of function resulted in late gestational lethality. Furthermore, hypertonicity-induced NFAT5/TonEBP transcriptional activity and hsp70.1 promoter function were completely eliminated, and cell proliferation under hyperosmotic culture conditions was markedly impaired. Partial loss of NFAT5/TonEBP function resulted in lymphoid hypocellularity and impaired antigen-specific antibody responses in viable heterozygous animals. In addition, lymphocyte proliferation ex vivo was reduced under hypertonic, but not isotonic, culture conditions. Direct measurement of tissue osmolality further revealed lymphoid tissues to be hyperosmolar. These results indicate that lymphocyte-mediated immunity is contingent on adaptation to physiologic osmotic stress, thus providing insight into the lymphoid microenvironment and the importance of the NFAT5/TonEBP osmotic stress response pathway in vivo.

PGs and leukotrienes (LTs) mediate cardinal signs of inflammation; hence, their enzymes are targets of current anti-inflammatory therapies. Products of arachidonate 15-lipoxygenases (LO) types I and II display both beneficial roles, such as lipoxins (LXs) that stereoselectively signal counterregulation, as well as potential deleterious actions (i.e., nonspecific phospholipid degradation). In this study, we examined transgenic (TG) rabbits overexpressing 15-LO type I and their response to inflammatory challenge. Skin challenges with either LTB(4) or IL-8 showed that 15-LO TG rabbits give markedly reduced neutrophil (PMN) recruitment and plasma leakage at dermal sites with LTB(4). PMN from TG rabbits also exhibited a dramatic reduction in LTB(4)-stimulated granular mobilization that was not evident with peptide chemoattractants. Leukocytes from 15-LO TG rabbits gave enhanced LX production, underscoring differences in lipid mediator profiles compared with non-TG rabbits. Microbe-associated inflammation and leukocyte-mediated bone destruction were assessed by initiating acute periodontitis. 15-LO TG rabbits exhibited markedly reduced bone loss and local inflammation. Because enhanced LX production was associated with an increased anti-inflammatory status of 15-LO TG rabbits, a stable analog of 5S,6R,15S-trihydroxyeicosa-7E,9E,11Z,13E-tetraenoic acid (LXA(4)) was applied to the gingival crevice subject to periodontitis. Topical application with the 15-epi-16-phenoxy-para-fluoro-LXA(4) stable analog (ATLa) dramatically reduced leukocyte infiltration, ensuing bone loss as well as inflammation. These results indicate that overexpression of 15-LO type I and LXA(4) is associated with dampened PMN-mediated tissue degradation and bone loss, suggesting that enhanced anti-inflammation status is an active process. Moreover, they suggest that LXs can be targets for novel approaches to diseases, e.g., periodontitis and arthritis, where inflammation and bone destruction are features.

The three-dimensional structure of the kinetochore and the DNA/protein composition of the centromere-kinetochore region was investigated using two novel techniques, caffeine-induced detachment of unreplicated kinetochores and stretching of kinetochores by hypotonic and/or shear forces generated in a cytocentrifuge. Kinetochore detachment was confirmed by EM and immunostaining with CREST autoantibodies. Electron microscopic analyses of serial sections demonstrated that detached kinetochores represented fragments derived from whole kinetochores. This was especially evident for the seven large kinetochores in the male Indian muntjac that gave rise to 80-100 fragments upon detachment. The kinetochore fragments, all of which interacted with spindle microtubules and progressed through the entire repertoire of mitotic movements, provide evidence for a subunit organization within the kinetochore. Further support for a repeat subunit model was obtained by stretching or uncoiling the metaphase centromere-kinetochore complex by hypotonic treatments. When immunostained with CREST autoantibodies and subsequently processed for in situ hybridization using synthetic centromere probes, stretched kinetochores displayed a linear array of fluorescent subunits arranged in a repetitive pattern along a centromeric DNA fiber. In addition to CREST antigens, each repetitive subunit was found to bind tubulin and contain cytoplasmic dynein, a microtubule motor localized in the zone of the corona. Collectively, the data suggest that the kinetochore, a plate-like structure seen by EM on many eukaryotic chromosomes is formed by the folding of a linear DNA fiber consisting of tandemly repeated subunits interspersed by DNA linkers. This model, unlike any previously proposed, can account for the structural and evolutional diversity of the kinetochore and its relationship to the centromere of eukaryotic chromosomes of many species.

Non-covalent residue side-chain interactions occur in many different types of proteins and facilitate many biological functions. Are these differences manifested in the sequence compositions and/or the residue-residue contact preferences of the interfaces? Previous studies analysed small data sets and gave contradictory answers. Here, we introduced a new data-mining method that yielded the largest high-resolution data set of interactions analysed. We introduced an information theory-based analysis method. On the basis of sequence features, we were able to differentiate six types of protein interfaces, each corresponding to a different functional or structural association between residues. Particularly, we found significant differences in amino acid composition and residue-residue preferences between interactions of residues within the same structural domain and between different domains, between permanent and transient interfaces, and between interactions associating homo-oligomers and hetero-oligomers. The differences between the six types were so substantial that, using amino acid composition alone, we could predict statistically to which of the six types of interfaces a pool of 1000 residues belongs at 63-100% accuracy. All interfaces differed significantly from the background of all residues in SWISS-PROT, from the group of surface residues, and from internal residues that were not involved in non-trivial interactions. Overall, our results suggest that the interface type could be predicted from sequence and that interface-type specific mean-field potentials may be adequate for certain applications.

Hyperpolarization-activated cation currents (I(h)) have been identified in cardiac pacemaker cells and a variety of central and peripheral neurons. Four members of a gene family encoding hyperpolarization-activated, cyclic nucleotide-gated cation channels (HCN1--4) have been cloned recently. Native I(h) currents recorded from different cell types exhibit distinct activation kinetics. To determine if this diversity of I(h) currents may be caused by differential expression of HCN channel isoforms, we investigated the cellular distribution of the transcripts of HCN1--4 in the murine sinoatrial node, retina and dorsal root ganglion (DRG) by in situ hybridization. In the sinoatrial node, the most prominently expressed HCN channel is HCN4, whereas HCN2 and HCN1 are detected there at moderate and low levels, respectively. Retinal photoreceptors express high levels of HCN1, whereas HCN2, 3 and 4 were not found in these cells. In DRG neurons, the dominant HCN transcript is HCN1, followed by HCN2. We next determined the functional properties of recombinant HCN1--4 channels expressed in HEK293 cells. All four channel types gave rise to I(h) currents but displayed marked differences in their activation kinetics. Our results suggest that the heterogeneity of native I(h) currents is generated, at least in part, by the tissue-specific expression of HCN channel genes.

Xyr1 (xylanase regulator 1) of the ascomycete Hypocrea jecorina (anamorph Trichoderma reesei) was recently demonstrated to play an essential role in the transcriptional regulation of the xyn1 (xylanase 1-encoding) gene expression. Consequently, this study reports on the deletion of the xyr1 gene from the H. jecorina genome. Comparative studies of the growth behavior of the different mutant strains (deleted and retransformed xyr1) grown on various carbon sources pointed to the strongly reduced ability of the xyr1 deletion strain to utilize D-xylose and xylan. Transcriptional analysis of the xyl1 (D-xylose reductase 1-encoding) gene as well as measurements of corresponding enzymatic activities gave evidence that Xyr1 takes part in the control of the fungal D-xylose pathway, in particular in the regulation of D-xylose reductase. It could be demonstrated that the uptake of D-xylose into the fungal cell is uninfluenced in the Deltaxyr1 strain. Furthermore, transcriptional regulation of the major hydrolytic enzyme-encoding genes xyn1 and xyn2 (xylanases 1 and 2), cbh1 and cbh2 (cellobiohydrolases 1 and 2), and egl1 (endoglucanase 1) is strictly dependent on Xyr1. Regulation of the respective genes via Xyr1 is not affected by the substances mediating induction (xylose, xylobiose, and sophorose) and is indispensable for all modes of gene expression (basal, derepressed, and induced). Moreover, Xyr1, it was revealed, activated transcriptional regulation of inducer-providing enzymes such as beta-xylosidase BXLI and beta-glucosidase BGLI but was not shown to be involved in the regulation of BGLII.

A novel penicillin-binding protein, PBP-2' (Mr about 75,000), is known to be induced in excessively large amount by most beta-lactam compounds in cells of a clinically isolated strain of Staphylococcus aureus, TK784, that is highly resistant to beta-lactams and also most other antibiotics. This protein has very low affinities to most beta-lactam compounds and has been supposed to be the cause of the resistance of the cells to beta-lactams. A 14-kilobase DNA fragment was isolated from the cells that carried the gene encoding this penicillin-binding protein and also a genetically linked marker that is responsible for the resistance to tobramycin. This DNA was cloned on plasmid pACYC184 and was shown to cause both production of PBP-2' and resistance to tobramycin in Escherichia coli cells. However, the formation of PBP-2' in E. coli was only moderate and was independent of normal inducer beta-lactams. The PBP-2' formed in the E. coli cells showed slow kinetics of binding to beta-lactams similar to that of PBP-2' formed in the original S. aureus cells and gave a similar pattern of peptides to the latter when digested with the proteolytic V8 enzyme of S. aureus.

BACKGROUND

PD-L1 and CTLA-4 immune checkpoints inhibit antitumour T-cell activity. Combination treatment with the anti-PD-L1 antibody durvalumab and the anti-CTLA-4 antibody tremelimumab might provide greater antitumour activity than either drug alone. We aimed to assess durvalumab plus tremelimumab in patients with advanced squamous or non-squamous non-small cell lung cancer (NSCLC).

METHODS

We did a multicentre, non-randomised, open-label, phase 1b study at five cancer centres in the USA. We enrolled immunotherapy-naive patients aged 18 years or older with confirmed locally advanced or metastatic NSCLC. We gave patients durvalumab in doses of 3 mg/kg, 10 mg/kg, 15 mg/kg, or 20 mg/kg every 4 weeks, or 10 mg/kg every 2 weeks, and tremelimumab in doses of 1 mg/kg, 3 mg/kg, or 10 mg/kg every 4 weeks for six doses then every 12 weeks for three doses. The primary endpoint of the dose-escalation phase was safety. Safety analyses were based on the as-treated population. The dose-expansion phase of the study is ongoing. This study is registered with ClinicalTrials.gov, number NCT02000947.

RESULTS

Between Oct 28, 2013, and April 1, 2015, 102 patients were enrolled into the dose-escalation phase and received treatment. At the time of this analysis (June 1, 2015), median follow-up was 18·8 weeks (IQR 11-33). The maximum tolerated dose was exceeded in the cohort receiving durvalumab 20 mg/kg every 4 weeks plus tremelimumab 3 mg/kg, with two (30%) of six patients having a dose-limiting toxicity (one grade 3 increased aspartate aminotransferase and alanine aminotransferase and one grade 4 increased lipase). The most frequent treatment-related grade 3 and 4 adverse events were diarrhoea (11 [11%]), colitis (nine [9%]), and increased lipase (eight [8%]). Discontinuations attributable to treatment-related adverse events occurred in 29 (28%) of 102 patients. Treatment-related serious adverse events occurred in 37 (36%) of 102 patients. 22 patients died during the study, and three deaths were related to treatment. The treatment-related deaths were due to complications arising from myasthenia gravis (durvalumab 10 mg/kg every 4 weeks plus tremelimumab 1 mg/kg), pericardial effusion (durvalumab 20 mg/kg every 4 weeks plus tremelimumab 1 mg/kg), and neuromuscular disorder (durvalumab 20 mg/kg every 4 weeks plus tremelimumab 3 mg/kg). Evidence of clinical activity was noted both in patients with PD-L1-positive tumours and in those with PD-L1-negative tumours. Investigator-reported confirmed objective responses were achieved by six (23%, 95% CI 9-44) of 26 patients in the combined tremelimumab 1 mg/kg cohort, comprising two (22%, 95% CI 3-60) of nine patients with PD-L1-positive tumours and four (29%, 95% CI 8-58) of 14 patients with PD-L1-negative tumours, including those with no PD-L1 staining (four [40%, 95% CI 12-74] of ten patients).

CONCLUSIONS

Durvalumab 20 mg/kg every 4 weeks plus tremelimumab 1 mg/kg showed a manageable tolerability profile, with antitumour activity irrespective of PD-L1 status, and was selected as the dose for phase 3 studies, which are ongoing.

BACKGROUND

MedImmune.

The first colonisation of the intestine is one of the most profound immunological exposures faced by the newborn and it is influenced by external and internal factors. The early composition of human microbiota could have long-lasting metabolic effects and the initial composition of human intestinal bacteria is also known to affect postnatal immune system development, as we are already aware that reduced microbial stimulation during infancy would result in slower postnatal maturation of the immune system and development of an optimal balance between TH1 and TH2-like immunity. Mode of delivery has a major role on the composition of intestinal microbiota in early infancy, as it has been shown that infants born by Caesarean section (CS) have lower numbers of Bifidobacteria and Bacteroides compared with vaginally born infants. We designed a study to investigate the influence of mode of delivery (CS vs. vaginal delivery) on intestinal microbial composition on day 3 of life using PCR-denaturing gradient gel electrophoresis (DGGE) and PCR-temperature gradient gel electrophoresis (TGGE). Both DGGE and TGGE analyses have been used, together with the specific amplifications for 10 Bifidobacterium sp., 3 Ruminococcus sp., and Bacteroides that all have a highly relevant physiological role in the intestinal ecosystem of the newborn. A total of 46 term infants were enrolled in the study, consecutively recruiting all the CS-delivered babies (n=23; 8 males and 15 females) and the immediately following spontaneously delivered babies (n=23; 11 males and 12 females). DGGE analysis carried out with Bifidobacterium-specific primers revealed the presence of this genus in 13 of 23 (56.5%) samples derived from vaginally delivered newborns but in none of the samples obtained from newborns delivered by CS. PCR analysis with Bifidobacterium-species-specific primers showed that naturally delivered infants had a large number of bifidobacterial species, whereas in CS-delivered babies only two samples (8.7%) gave positive results, one for B. longum and another for B. gallicum. In all babies enrolled, micro-organisms belonging to Ruminococcus species were absent and Bacteroides was found in 8.7% of spontaneously delivered babies only. Based on our findings, it seems that newborn's intestinal bacteria during the first 3days of life are strongly influenced by mode of delivery. The intestinal flora of CS and vaginally delivered infants appears to be very different; the former being altered and characterised by a substantial absence of Bifidobacteria sp., the latter characterised by subject-specific microbial profiles, although predominant groups such as B. longum and B. catenulatum could be identified. In summary, mode of delivery does affect the early stage of intestinal bacterial colonisation, which is altered in CS-delivered infants compared with vaginally delivered infants, with only a minor influence of the type of feeding. In addition, the importance of methodological aspects for determining intestinal microbiota in clinical trials requires emphasis if intestinal microbiota composition is to be considered a measure of postnatal adaptation.

Borrelia burgdorferi strains (n = 23), isolated from patients (n = 8) and ticks (n = 14), were analyzed by SDS PAGE and Western blotting with monoclonal antibodies and polyclonal sera (rabbit immune serum and sera from patients). Testing the 23 strains by SDS PAGE 9 different patterns of major protein bands were observed. In contrast to US strains some of our strains showed only weak or negative reactivity with the OspA specific monoclonal antibody H5332. Analysis with polyclonal sera gave further support that the OspA proteins of our strains have specific epitopes (recognized by patients sera) besides common ones (recognized by rabbit immune serum). Fifteen of the strains had another major protein in the 22K range without detectable antigenic variability. Patients with erythema migrans and lymphocytic meningitis had a good IgM- and IgG-immunoresponse to this protein. In contrast antibodies to the OspA were very rarely observed, probably due to the antigenetic heterogeneity of the causative agent on the one hand but also due to low response of the human immune system to the OspA on the other hand.

There are at least three possibilities for encoding information in a small area of cortex. First, neurons could have identical characteristics, thus conveying redundant information; second, neurons could give different responses to the same stimuli, thus conveying independent information; or third, neurons could cooperate with each other to encode more information jointly than they do separately, that is, synergistically. We recorded from 28 pairs of neurons in inferior temporal cortex of behaving rhesus monkeys. Each pair was recorded from a single microelectrode. Both the magnitude and the temporal modulation of the responses were quantified. We separated the responses into signal (average response to each stimulus) and noise (deviation of each response from the average). Linear regression showed that an average of only 18.7% of the magnitude of the signal carried by one neuron could be predicted from the magnitude of the other, and only 22.0% could be predicted by including the temporal modulation. For the noise, the figures were 5.5% and 6.3%, respectively, even less than for the signal. Information theoretic analysis shows that the pairs of neurons we studied carried an average of 20% redundant information. However, even this relatively small amount of redundancy places a severe upper limit on the information that can be transmitted by a neuronal pool. A pool of neurons for which each pair is mutually redundant to extent y can only carry a maximum of 1/y, here five times, as much information as one neuron alone. Information theoretic analysis gave no evidence for the presence of information as a function of both neurons considered together, that is, synergistic codes. Cross-correlation showed that at least 61% of the neuronal pairs shared connections in some manner. Given these shared connections, if adjacent neurons had had identical characteristics, then the noise on the outputs of these neurons would have been highly correlated, and it would not be possible to separate the signal and noise. The severe impact of correlated noise and information redundancy leads us to propose that the processing carried out by these neurons evolved both to provide a rich description of many stimulus properties and simultaneously to minimize the redundancy in a local group of neurons. These two principles appear to be a major constraint on the organization of inferior temporal, and possibly all, cortex.

An experimental vaccine that was based on secreted proteins of Mycobacterium tuberculosis was investigated in a mouse model of tuberculosis. I used a short-term culture filtrate (ST-CF) containing proteins secreted from actively replicating bacteria grown under defined culture conditions. The immunogenicity of the ST-CF was investigated in combination with different adjuvants, and peak proliferative responses were observed when ST-CF was administered with the surface-active agent dimethyldioctadecylammonium chloride. The immunity induced by this vaccine was dose dependent, and, in the optimal concentration, the vaccine induced a potent T-helper 1 response which efficiently protected the animals against a subsequent challenge with virulent M. tuberculosis. Antigenic targets for the T cells generated were mapped by employing narrow-molecular-weight fractions of ST-CF. The experimental vaccine primed a broadly defined T-cell repertoire directed to multiple secreted antigens present in ST-CF. A vaccination with viable Mycobacterium bovis bacillus Calmette-Guérin (BCG), in contrast, induced a restricted T-cell reactivity directed to two secreted protein fractions with molecular masses of 5 to 12 and 25 to 35 kDa. The protective efficacy of the ST-CF vaccine was compared with that of a BCG standard vaccine, and both induced a highly significant protection of equal magnitude. The vaccination with ST-CF gave rise to a population of long-lived CD4 cells which could be isolated 22 weeks after the vaccination and could adoptively transfer acquired resistance to T-cell-deficient recipients. My results confirm the hypothesis that M. tuberculosis cells release protective antigens during growth. The high efficacy of a subunit vaccine observed in the present study is discussed as a possible alternative to a live recombinant vaccine carrier.

1. Xanthine oxidase acting aerobically upon acetaldehyde was found to cause the peroxidation of linolenate. This was demonstrated by increased absorbance at 233 nm due to diene conjugation and by the detection of a lipid peroxide spot on the thin layer chromatograms. 2. Superoxide dismutase inhibited this lipid peroxidation, as did catalase, thus indicating that both O2- and H2O2 were essential intermediates. Scavengers of singlet oxygen also inhibited the peroxidation of linolenate, whereas scavengers of hydroxyl radical did not. These effects, which were observed in the absence of iron salts, led to the proposal that O2- and H2O2 can directly give rise to a singlet oxygen, as follows: O2- + H2O2 leads to OH- + OH. + O2. 3. This proposal was further supported through the use of 2,5-dimethylfuran, as an indicating scavenger of singlet oxygen. Thus, when this compound was exposed to a known source of singlet oxygen, it gave a product which was detectable by thin layer chromatography. This product was also observed when 2,5-dimethylfuran was exposed to the xanthine oxidase system, in which case its accumulation was prevented by superoxide dismutase or by catalase, but not by scavengers of hydroxyl radical.

OBJECTIVE

To compare perinatal outcomes, maternal outcomes, and interventions in labour by planned place of birth at the start of care in labour for women with low risk pregnancies.

METHODS

Prospective cohort study.

METHODS

England: all NHS trusts providing intrapartum care at home, all freestanding midwifery units, all alongside midwifery units (midwife led units on a hospital site with an obstetric unit), and a stratified random sample of obstetric units.

METHODS

64,538 eligible women with a singleton, term (≥37 weeks gestation), and "booked" pregnancy who gave birth between April 2008 and April 2010. Planned caesarean sections and caesarean sections before the onset of labour and unplanned home births were excluded.

METHODS

A composite primary outcome of perinatal mortality and intrapartum related neonatal morbidities (stillbirth after start of care in labour, early neonatal death, neonatal encephalopathy, meconium aspiration syndrome, brachial plexus injury, fractured humerus, or fractured clavicle) was used to compare outcomes by planned place of birth at the start of care in labour (at home, freestanding midwifery units, alongside midwifery units, and obstetric units).

RESULTS

There were 250 primary outcome events and an overall weighted incidence of 4.3 per 1000 births (95% CI 3.3 to 5.5). Overall, there were no significant differences in the adjusted odds of the primary outcome for any of the non-obstetric unit settings compared with obstetric units. For nulliparous women, the odds of the primary outcome were higher for planned home births (adjusted odds ratio 1.75, 95% CI 1.07 to 2.86) but not for either midwifery unit setting. For multiparous women, there were no significant differences in the incidence of the primary outcome by planned place of birth. Interventions during labour were substantially lower in all non-obstetric unit settings. Transfers from non-obstetric unit settings were more frequent for nulliparous women (36% to 45%) than for multiparous women (9% to 13%).

CONCLUSIONS

The results support a policy of offering healthy women with low risk pregnancies a choice of birth setting. Women planning birth in a midwifery unit and multiparous women planning birth at home experience fewer interventions than those planning birth in an obstetric unit with no impact on perinatal outcomes. For nulliparous women, planned home births also have fewer interventions but have poorer perinatal outcomes.

We report herein the application of a set of algorithms to identify a large number (2869) of single-copy orthologs (COSII), which are shared by most, if not all, euasterid plant species as well as the model species Arabidopsis. Alignments of the orthologous sequences across multiple species enabled the design of "universal PCR primers," which can be used to amplify the corresponding orthologs from a broad range of taxa, including those lacking any sequence databases. Functional annotation revealed that these conserved, single-copy orthologs encode a higher-than-expected frequency of proteins transported and utilized in organelles and a paucity of proteins associated with cell walls, protein kinases, transcription factors, and signal transduction. The enabling power of this new ortholog resource was demonstrated in phylogenetic studies, as well as in comparative mapping across the plant families tomato (family Solanaceae) and coffee (family Rubiaceae). The combined results of these studies provide compelling evidence that (1) the ancestral species that gave rise to the core euasterid families Solanaceae and Rubiaceae had a basic chromosome number of x=11 or 12.2) No whole-genome duplication event (i.e., polyploidization) occurred immediately prior to or after the radiation of either Solanaceae or Rubiaceae as has been recently suggested.

The origin, roles and fate of progenitor cells forming synovial joints during limb skeletogenesis remain largely unclear. Here we produced prenatal and postnatal genetic cell fate-maps by mating ROSA-LacZ-reporter mice with mice expressing Cre-recombinase at prospective joint sites under the control of Gdf5 regulatory sequences (Gdf5-Cre). Reporter-expressing cells initially constituted the interzone, a compact mesenchymal structure representing the first overt sign of joint formation, and displayed a gradient-like distribution along the ventral-to-dorsal axis. The cells expressed genes such as Wnt9a, Erg and collagen IIA, remained predominant in the joint-forming sites over time, gave rise to articular cartilage, synovial lining and other joint tissues, but contributed little if any to underlying growth plate cartilage and shaft. To study their developmental properties more directly, we isolated the joint-forming cells from prospective autopod joint sites using a novel microsurgical procedure and tested them in vitro. The cells displayed a propensity to undergo chondrogenesis that was enhanced by treatment with exogenous rGdf5 but blocked by Wnt9a over-expression. To test roles for such Wnt-mediated anti-chondrogenic capacity in vivo, we created conditional mutants deficient in Wnt/beta-catenin signaling using Col2-Cre or Gdf5-Cre. Synovial joints did form in both mutants; however, the joints displayed a defective flat cell layer normally abutting the synovial cavity and expressed markedly reduced levels of lubricin. In sum, our data indicate that cells present at prospective joint sites and expressing Gdf5 constitute a distinct cohort of progenitor cells responsible for limb joint formation. The cells appear to be patterned along specific limb symmetry axes and rely on local signaling tools to make distinct contributions to joint formation.

Bacterial identification relies primarily on culture-based methodologies requiring 24 h for isolation and an additional 24 to 48 h for species identification. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) is an emerging technology newly applied to the problem of bacterial species identification. We evaluated two MALDI-TOF MS systems with 720 consecutively isolated bacterial colonies under routine clinical laboratory conditions. Isolates were analyzed in parallel on both devices, using the manufacturers' default recommendations. We compared MS with conventional biochemical test system identifications. Discordant results were resolved with "gold standard" 16S rRNA gene sequencing. The first MS system (Bruker) gave high-confidence identifications for 680 isolates, of which 674 (99.1%) were correct; the second MS system (Shimadzu) gave high-confidence identifications for 639 isolates, of which 635 (99.4%) were correct. Had MS been used for initial testing and biochemical identification used only in the absence of high-confidence MS identifications, the laboratory would have saved approximately US$5 per isolate in marginal costs and reduced average turnaround time by more than an 8-h shift, with no loss in accuracy. Our data suggest that implementation of MS as a first test strategy for one-step species identification would improve timeliness and reduce isolate identification costs in clinical bacteriology laboratories now.