Studies of the identity and physiological function of mesenchymal stromal cells (MSCs) have been hampered by a lack of markers that permit both prospective identification and fate mapping in vivo. We found that Leptin Receptor (LepR) is a marker that highly enriches bone marrow MSCs. Approximately 0.3% of bone marrow cells were LepR(+), 10% of which were CFU-Fs, accounting for 94% of bone marrow CFU-Fs. LepR(+) cells formed bone, cartilage, and adipocytes in culture and upon transplantation in vivo. LepR(+) cells were Scf-GFP(+), Cxcl12-DsRed(high), and Nestin-GFP(low), markers which also highly enriched CFU-Fs, but negative for Nestin-CreER and NG2-CreER, markers which were unlikely to be found in CFU-Fs. Fate-mapping showed that LepR(+) cells arose postnatally and gave rise to most bone and adipocytes formed in adult bone marrow, including bone regenerated after irradiation or fracture. LepR(+) cells were quiescent, but they proliferated after injury. Therefore, LepR(+) cells are the major source of bone and adipocytes in adult bone marrow.

We have devised a method for detecting and estimating the sizes of large bacterial plasmids in the presence of genomic DNA by pulsed-field gel electrophoresis (PFGE). Bacteria harboring plasmids were embedded in agarose and lysed using a rapid protocol. Plugs were incubated with S1 nuclease and subjected to PFGE in agarose gels. S1 nuclease converted supercoiled plasmids into full-length linear molecules. Large plasmids migrated as discrete bands that were readily observed after ethidium staining. Their sizes were reliably estimated by comparison with linear DNA markers. Without S1 digestion, supercoiled plasmids migrated at rates that were not a simple function of their molecular weights, making size determinations problematic. S1-PFGE detected megaplasmids up to 609 kilobases (kb) in six genera of bacteria (Agrobacterium, Escherichia, Klebsiella, Pseudomonas, Salmonella, and Staphylococcus). The procedure gave size values consistent with previous estimates for characterized megaplasmids. Eight new plasmids between 102 and 316 kb were discovered in Klebsiella and Staphylococcus. S1-PFGE avoids the difficulties of plasmid isolation, eliminates the preparation of probes, and does not require knowledge of restriction enzyme cleavage sites. It detects multiple large plasmids up to the limits of PFGE and can be used to screen for megaplasmids in many strains simultaneously.

BACKGROUND

A farming environment protects against development of asthma, hay fever, and atopic sensitisation in children. We aimed to establish whether increased exposure to microbial compounds has to occur early in life to affect maturation of the immune system and thereby reduces risk for development of allergic diseases.

METHODS

We did a cross-sectional survey in rural areas of Austria, Germany, and Switzerland. 2618 (75%) of 3504 parents of 6-13-year-old children completed a standardised questionnaire on asthma, hay fever, and atopic eczema. Children from farming families, and a random sample of non-farmers' children, who gave consent for blood samples to be obtained for measurements of specific serum IgE antibodies to common allergens were invited to participate (n=901).

RESULTS

Exposure of children younger than 1 year, compared with those aged 1-5 years, to stables and consumption of farm milk was associated with lower frequencies of asthma (1% [3/218] vs 11% [15/138]), hay fever (3% [7] vs 13% [18]), and atopic sensitisation (12% [27] vs 29% [40]). Protection against development of asthma was independent from effect on atopic sensitisation. Continual long-term exposure to stables until age 5 years was associated with the lowest frequencies of asthma (0.8% [1/122]), hay fever (0.8% [1]), and atopic sensitisation (8.2% [10]).

CONCLUSIONS

Long-term and early-life exposure to stables and farm milk induces a strong protective effect against development of asthma, hay fever, and atopic sensitisation.

A routing radioimmunoassay for human C-peptide in serum is described. Antibodies against human C-peptide were raised by immunizing guinea pigs with human b-component. Nine out of 12 animals produced useful antibodies within 6 months. Insulin antibodies coupled to Sepharose were used to bind human proinsulin and insulin in the serum and after centrifugation C-peptide was determined in the supernatant. The detection limit of the assay (calculated as 2 SD from zero) was about 0.003 pmole of C-peptide (in 100 mul). The main sources of error were: (1) Normal and diabetic sera devoid of C-peptide gave a displacement of 125I-Tyr-C-peptide varying from 0 to 0.16 nM (6 different antisera). Only one antiserum (M 1181) showed no displacement, and the values of C-peptide determined with this antiserum in normal and diabetic sera were lower than the values determined with another antiserum, which gave a value of 0.07 nM in the sera free of C-peptide. It is suggested that displacement found with most antisera is due to substances in serum that are not related to C-peptide or proinsulin. (2) Serial dilutions of pancreatic extracts and sera may yield dilution curves slightly different to those of the synthetic standard. Possible explanations are discussed. These sources of error can be eliminated or reduced by the proper selection of antisera. Fasting sera from 15 normals, 8 maturity-onset diabetics and 10 insulin-requiring diabetics showed the following concentrations of C-peptide: (M 1181) 0.35 +/- 0.09, 0.74 +/- 0.51 and 0.21 +/- 0.14 (nM, mean +/- SD). One hour after 1.75 g/kg oral glucose the values increased to 2.24 +/- 0.71, 2.34 +/- 0.24 nM.

Cultures of plastic-adherent cells from bone marrow have attracted interest because of their ability to support growth of hematopoietic stem cells, their multipotentiality for differentiation, and their possible use for cell and gene therapy. Here we found that the cells grew most rapidly when they were initially plated at low densities (1.5 or 3.0 cells/cm(2)) to generate single-cell derived colonies. The cultures displayed a lag phase of about 5 days, a log phase of rapid growth of about 5 days, and then a stationary phase. FACS analysis demonstrated that stationary cultures contained a major population of large and moderately granular cells and a minor population of small and agranular cells here referred to as recycling stem cells or RS-1 cells. During the lag phase, the RS-1 cells gave rise to a new population of small and densely granular cells (RS-2 cells). During the late log phase, the RS-2 cells decreased in number and regenerated the pool of RS-1 cells found in stationary cultures. In repeated passages in which the cells were plated at low density, they were amplified about 10(9)-fold in 6 wk. The cells retained their ability to generate single-cell derived colonies and therefore apparently retained their multipotentiality for differentiation.

Acinetobacter baumannii is a species of nonfermentative gram-negative bacteria commonly found in water and soil. This organism was susceptible to most antibiotics in the 1970s. It has now become a major cause of hospital-acquired infections worldwide due to its remarkable propensity to rapidly acquire resistance determinants to a wide range of antibacterial agents. Here we use a comparative genomic approach to identify the complete repertoire of resistance genes exhibited by the multidrug-resistant A. baumannii strain AYE, which is epidemic in France, as well as to investigate the mechanisms of their acquisition by comparison with the fully susceptible A. baumannii strain SDF, which is associated with human body lice. The assembly of the whole shotgun genome sequences of the strains AYE and SDF gave an estimated size of 3.9 and 3.2 Mb, respectively. A. baumannii strain AYE exhibits an 86-kb genomic region termed a resistance island--the largest identified to date--in which 45 resistance genes are clustered. At the homologous location, the SDF strain exhibits a 20 kb-genomic island flanked by transposases but devoid of resistance markers. Such a switching genomic structure might be a hotspot that could explain the rapid acquisition of resistance markers under antimicrobial pressure. Sequence similarity and phylogenetic analyses confirm that most of the resistance genes found in the A. baumannii strain AYE have been recently acquired from bacteria of the genera Pseudomonas, Salmonella, or Escherichia. This study also resulted in the discovery of 19 new putative resistance genes. Whole-genome sequencing appears to be a fast and efficient approach to the exhaustive identification of resistance genes in epidemic infectious agents of clinical significance.

We have determined the complete DNA sequence of the short unique region in the genome of herpes simplex virus type 1, strain 17, and have interpreted it in terms of messenger RNAs and encoded proteins. The sequence contains variable regions whose length differs between DNA clones. The clones used for most of the analysis gave a short unique length of 12,979 base-pairs. We consider that this region contains 12 genes, which are expressed by mRNAs which have separate promoters, but may share 3'-termination sites, so that all but two mRNAs belong to one of four 3'-coterminal "families": 79% of the sequence is considered to be polypeptide coding. One pair of genes has an extensive out-of-frame overlap of coding sequences. The proteins encoded in the short unique region include two immediate-early species, two virion surface glycoproteins, and a DNA-binding species. Six of the genes have little or no previous characterization. From the nature of the amino acid sequences predicted for their encoded proteins, we deduce that several of these proteins may be membrane-associated.

Trimeric and tetrameric short tandem repeats (STRs) represent a rich source of highly polymorphic markers in the human genome that may be studied with the polymerase chain reaction (PCR). We report the analysis of a multilocus genotype survey of 97-380 chromosomes in U.S. Black, White, Mexican-American, and Asian populations at five STR loci located on chromosomes 1, 4, 11, and X. The heterozygote frequencies of the loci ranged from 0.36 to 0.91 and the number of alleles from 6 to 20 for the 20 population and locus combinations. Relative allele frequencies exhibited differences between populations and unimodal, bimodal, and complex distributions. Although deviations were noted at some locus-population test combinations, genotype data from the loci were consistent overall with Hardy-Weinberg equilibrium by three tests. Population subheterogeneity within each ethnic group was not detected by two additional tests. No mutations were detected in a total of 860 meioses for two loci studied in the CEPH kindreds and five loci studied in other families. An indirect estimate of the mutation rates gave values from 2.3 x 10(-5) to 15.9 x 10(-5) for the five loci. Higher mutation rates appear to be associated with greater numbers of tandem repeats in the core motif. The most frequent genotype for all five loci combined appears to have a frequency of 7.59 x 10(-4). Together, these results suggest that trimeric and tetrameric STR loci are useful markers for the study of new mutations and genetic linkage analysis and for application to personal identification in the medical and forensic sciences.

The importance of body fat distribution as a predictor of metabolic aberrations was evaluated in 9 nonobese and 25 obese, apparently healthy women. Plasma glucose and insulin levels during oral glucose loading were significantly higher in women with predominantly upper body segment obesity than in women with lower body segment obesity. Of the former group, 10 of 16 subjects had diabetic glucose tolerance results, while none of the latter group was diabetic. Fasting plasma triglyceride levels were also significantly higher in the upper body segment obese women. The site of adiposity in the upper body segment obese women was comprised of large fat cells, while in the lower body segment obese subjects, it was formed of normal size cells. In both types of obesity, abdominal fat cell size correlated significantly with postprandial plasma glucose and insulin levels. Thigh fat cell size gave no indication as to the presence of metabolic complications. Thigh adipocytes were also resistant to epinephrine-stimulated lipolysis, presumably due to an increase in alpha-adrenergic receptors. Thus, in women, the sites of fat predominance offer an important prognostic marker for glucose intolerance, hyperinsulinemia, and hypertriglyceridemia. This association may be related to the disparate morphology and metabolic behavior of fat cells associated with different body fat distributions.

OBJECTIVE

To compare the performance of different meta-analysis methods for pooling odds ratios when applied to sparse event data with emphasis on the use of continuity corrections.

BACKGROUND

Meta-analysis of side effects from RCTs or risk factors for rare diseases in epidemiological studies frequently requires the synthesis of data with sparse event rates. Combining such data can be problematic when zero events exist in one or both arms of a study as continuity corrections are often needed, but, these can influence results and conclusions.

METHODS

A simulation study was undertaken comparing several meta-analysis methods for combining odds ratios (using various classical and Bayesian methods of estimation) on sparse event data. Where required, the routine use of a constant and two alternative continuity corrections; one based on a function of the reciprocal of the opposite group arm size; and the other an empirical estimate of the pooled effect size from the remaining studies in the meta-analysis, were also compared. A number of meta-analysis scenarios were simulated and replicated 1000 times, varying the ratio of the study arm sizes.

RESULTS

Mantel-Haenszel summary estimates using the alternative continuity correction factors gave the least biased results for all group size imbalances. Logistic regression was virtually unbiased for all scenarios and gave good coverage properties. The Peto method provided unbiased results for balanced treatment groups but bias increased with the ratio of the study arm sizes. The Bayesian fixed effect model provided good coverage for all group size imbalances. The two alternative continuity corrections outperformed the constant correction factor in nearly all situations. The inverse variance method performed consistently badly, irrespective of the continuity correction used.

CONCLUSIONS

Many routinely used summary methods provide widely ranging estimates when applied to sparse data with high imbalance between the size of the studies' arms. A sensitivity analysis using several methods and continuity correction factors is advocated for routine practice.

The mechanism by which cholera toxin (CT) is internalized from the plasma membrane before its intracellular reduction and subsequent activation of adenylyl cyclase is not well understood. Ganglioside GM1, the receptor for CT, is predominantly clustered in detergent-insoluble glycolipid rafts and in caveolae, noncoated, cholesterol-rich invaginations on the plasma membrane. In this study, we used filipin, a sterol-binding agent that disrupts caveolae and caveolae-like structures, to explore their role in the internalization and activation of CT in CaCo-2 human intestinal epithelial cells. When toxin internalization was quantified, only 33% of surface-bound toxin was internalized by filipin-treated cells within 1 h compared with 79% in untreated cells. However, CT activation as determined by its reduction to form the A1 peptide and CT activity as measured by cyclic AMP accumulation were inhibited in filipin-treated cells. Another sterol-binding agent, 2-hydroxy-beta-cyclodextrin, gave comparable results. The cationic amphiphilic drug chlorpromazine, an inhibitor of clathrin-dependent, receptor-mediated endocytosis, however, affected neither CT internalization, activation, nor activity in contrast to its inhibitory effects on diphtheria toxin cytotoxicity. As filipin did not inhibit the latter, the two drugs appeared to distinguish between caveolae- and coated pit-mediated processes. In addition to its effects in CaCo-2 cells that express low levels of caveolin, filipin also inhibited CT activity in human epidermoid carcinoma A431 and Jurkat T lymphoma cells that are, respectively, rich in or lack caveolin. Thus, filipin inhibition correlated more closely with alterations in the biochemical characteristics of CT-bound membranes due to the interactions of filipin with cholesterol rather than with the expressed levels of caveolin and caveolar structure. Our results indicated that the internalization and activation of CT was dependent on and mediated through cholesterol- and glycolipid-rich microdomains at the plasma membrane rather than through a specific morphological structure and that these glycolipid microdomains have the necessary components required to mediate endocytosis.

OBJECTIVE

To compare clinical, immunohistochemical (IHC), and gene expression models of prognosis applicable to formalin-fixed, paraffin-embedded blocks in a large series of estrogen receptor (ER)-positive breast cancers from patients uniformly treated with adjuvant tamoxifen.

METHODS

Quantitative real-time reverse transcription-PCR (qRT-PCR) assays for 50 genes identifying intrinsic breast cancer subtypes were completed on 786 specimens linked to clinical (median follow-up, 11.7 years) and IHC [ER, progesterone receptor (PR), HER2, and Ki67] data. Performance of predefined intrinsic subtype and risk-of-relapse scores was assessed using multivariable Cox models and Kaplan-Meier analysis. Harrell's C-index was used to compare fixed models trained in independent data sets, including proliferation signatures.

RESULTS

Despite clinical ER positivity, 10% of cases were assigned to nonluminal subtypes. qRT-PCR signatures for proliferation genes gave more prognostic information than clinical assays for hormone receptors or Ki67. In Cox models incorporating standard prognostic variables, hazard ratios for breast cancer disease-specific survival over the first 5 years of follow-up, relative to the most common luminal A subtype, are 1.99 [95% confidence interval (CI), 1.09-3.64] for luminal B, 3.65 (95% CI, 1.64-8.16) for HER2-enriched subtype, and 17.71 (95% CI, 1.71-183.33) for the basal-like subtype. For node-negative disease, PAM50 qRT-PCR-based risk assignment weighted for tumor size and proliferation identifies a group with >95% 10-year survival without chemotherapy. In node-positive disease, PAM50-based prognostic models were also superior.

CONCLUSIONS

The PAM50 gene expression test for intrinsic biological subtype can be applied to large series of formalin-fixed, paraffin-embedded breast cancers, and gives more prognostic information than clinical factors and IHC using standard cut points.

A new connective tissue protein, which we call fibrillin, has been isolated from the medium of human fibroblast cell cultures. Electrophoresis of the disulfide bond-reduced protein gave a single band with an estimated molecular mass of 350,000 D. This 350-kD protein appeared to possess intrachain disulfide bonds. It could be stained with periodic acid-Schiff reagent, and after metabolic labeling, it contained [3H]glucosamine. It could not be labeled with [35S]sulfate. It was resistant to digestion by bacterial collagenase. Using mAbs specific for fibrillin, we demonstrated its widespread distribution in the connective tissue matrices of skin, lung, kidney, vasculature, cartilage, tendon, muscle, cornea, and ciliary zonule. Electron microscopic immunolocalization with colloidal gold conjugates specified its location to a class of extracellular structural elements described as microfibrils. These microfibrils possessed a characteristic appearance and averaged 10 nm in diameter. Microfibrils around the amorphous cores of the elastic fiber system as well as bundles of microfibrils without elastin cores were labeled equally well with antibody. Immunolocalization suggested that fibrillin is arrayed periodically along the individual microfibril and that individual microfibrils may be aligned within bundles. The periodicity of the epitope appeared to match the interstitial collagen band periodicity. In contrast, type VI collagen, which has been proposed as a possible microfibrillar component, was immunolocalized with a specific mAb to small diameter microfilaments that interweave among the large, banded collagen fibers; it was not associated with the system of microfibrils identified by the presence of fibrillin.

The human body contains over 500 individual lymph nodes, yet the biology of their formation is poorly understood. Here we identify human lymphoid tissue-inducer cells (LTi cells) as lineage-negative RORC+ CD127+ cells with the functional ability to interact with mesenchymal cells through lymphotoxin and tumor necrosis factor. Human LTi cells were committed natural killer (NK) cell precursors that produced interleukin 17 (IL-17) and IL-22. In vitro, LTi cells gave rise to RORC+ CD127+ NK cells that retained the ability to produce IL-17 and IL-22. Postnatally, similar populations of LTi cell-like cells and RORC+ CD127+ NK cells were present in tonsils, and both secreted IL-17 and IL-22 but no interferon-gamma. Our data indicate that lymph node organogenesis is controlled by an NK cell precursor population with adaptive immune features and demonstrate a previously unappreciated link between the innate and adaptive immune systems.

Acquired immunodeficiency syndrome (AIDS) of humans is caused by two lentiviruses, human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2). Here, we describe the origins and evolution of these viruses, and the circumstances that led to the AIDS pandemic. Both HIVs are the result of multiple cross-species transmissions of simian immunodeficiency viruses (SIVs) naturally infecting African primates. Most of these transfers resulted in viruses that spread in humans to only a limited extent. However, one transmission event, involving SIVcpz from chimpanzees in southeastern Cameroon, gave rise to HIV-1 group M-the principal cause of the AIDS pandemic. We discuss how host restriction factors have shaped the emergence of new SIV zoonoses by imposing adaptive hurdles to cross-species transmission and/or secondary spread. We also show that AIDS has likely afflicted chimpanzees long before the emergence of HIV. Tracing the genetic changes that occurred as SIVs crossed from monkeys to apes and from apes to humans provides a new framework to examine the requirements of successful host switches and to gauge future zoonotic risk.

We report on the construction of a full-length cDNA clone of Semliki Forest virus (SFV). By placing the cDNA under the SP6 promoter, infectious RNA can be produced in vitro and used to transfect cells to initiate virus infection. To achieve efficient transfections, a new protocol for electroporation of RNA was developed. This method gave up to 500-fold improvement over the traditional DEAE-dextran transfection procedure. Since virtually 100% of the cells can be transfected by electroporation, this method is a useful tool for detailed biochemical studies of null mutations of SFV that abolish production of infections virus particles. We used the cDNA clone of SFV to study what effects a deletion of the 6,000-molecular-weight membrane protein (6K membrane protein) had on virus replication. The small 6K protein is part of the structural precursor molecule (C-p62-6K-E1) of the virus. Our results conclusively show that the 6K protein is not needed for the heterodimerization of the p62 and E1 spike membrane proteins in the endoplasmic reticulum, nor is it needed for their transport out to the cell surface. The absence of the 6K protein did, however, result in a dramatic reduction in virus release, suggesting that the protein exerts its function late in the assembly pathway, possibly during virus budding.

Cornelia de Lange syndrome (CdLS; OMIM 122470) is a dominantly inherited multisystem developmental disorder characterized by growth and cognitive retardation; abnormalities of the upper limbs; gastroesophageal dysfunction; cardiac, ophthalmologic and genitourinary anomalies; hirsutism; and characteristic facial features. Genital anomalies, pyloric stenosis, congenital diaphragmatic hernias, cardiac septal defects, hearing loss and autistic and self-injurious tendencies also frequently occur. Prevalence is estimated to be as high as 1 in 10,000 (ref. 4). We carried out genome-wide linkage exclusion analysis in 12 families with CdLS and identified four candidate regions, of which chromosome 5p13.1 gave the highest multipoint lod score of 2.7. This information, together with the previous identification of a child with CdLS with a de novo t(5;13)(p13.1;q12.1) translocation, allowed delineation of a 1.1-Mb critical region on chromosome 5 for the gene mutated in CdLS. We identified mutations in one gene in this region, which we named NIPBL, in four sporadic and two familial cases of CdLS. We characterized the genomic structure of NIPBL and found that it is widely expressed in fetal and adult tissues. The fly homolog of NIPBL, Nipped-B, facilitates enhancer-promoter communication and regulates Notch signaling and other developmental pathways in Drosophila melanogaster.

BACKGROUND

Observational studies report reduced colorectal cancer in regular aspirin consumers. Randomised controlled trials have shown reduced risk of adenomas but none have employed prevention of colorectal cancer as a primary endpoint. The CAPP2 trial aimed to investigate the antineoplastic effects of aspirin and a resistant starch in carriers of Lynch syndrome, the major form of hereditary colorectal cancer; we now report long-term follow-up of participants randomly assigned to aspirin or placebo.

METHODS

In the CAPP2 randomised trial, carriers of Lynch syndrome were randomly assigned in a two-by-two factorial design to 600 mg aspirin or aspirin placebo or 30 g resistant starch or starch placebo, for up to 4 years. Randomisation was in blocks of 16 with provision for optional single-agent randomisation and extended postintervention double-blind follow-up; participants and investigators were masked to treatment allocation. The primary endpoint was development of colorectal cancer. Analysis was by intention to treat and per protocol. This trial is registered, ISRCTN59521990.

RESULTS

861 participants were randomly assigned to aspirin or aspirin placebo. At a mean follow-up of 55·7 months, 48 participants had developed 53 primary colorectal cancers (18 of 427 randomly assigned to aspirin, 30 of 434 to aspirin placebo). Intention-to-treat analysis of time to first colorectal cancer showed a hazard ratio (HR) of 0·63 (95% CI 0·35-1·13, p=0·12). Poisson regression taking account of multiple primary events gave an incidence rate ratio (IRR) of 0·56 (95% CI 0·32-0·99, p=0·05). For participants completing 2 years of intervention (258 aspirin, 250 aspirin placebo), per-protocol analysis yielded an HR of 0·41 (0·19-0·86, p=0·02) and an IRR of 0·37 (0·18-0·78, p=0·008). No data for adverse events were available postintervention; during the intervention, adverse events did not differ between aspirin and placebo groups.

CONCLUSIONS

600 mg aspirin per day for a mean of 25 months substantially reduced cancer incidence after 55·7 months in carriers of hereditary colorectal cancer. Further studies are needed to establish the optimum dose and duration of aspirin treatment.

BACKGROUND

European Union; Cancer Research UK; Bayer Corporation; National Starch and Chemical Co; UK Medical Research Council; Newcastle Hospitals trustees; Cancer Council of Victoria Australia; THRIPP South Africa; The Finnish Cancer Foundation; SIAK Switzerland; Bayer Pharma.

Fusions of the secreted protein alkaline phosphatase to an integral cytoplasmic membrane protein of Escherichia coli showed different activities depending on where in the membrane protein the alkaline phosphatase was fused. Fusions to positions in or near the periplasmic domain led to high alkaline phosphatase activity, whereas those to positions in the cytoplasmic domain gave low activity. Analysis of alkaline phosphatase fusions to membrane proteins of unknown structure may thus be generally useful in determining their membrane topologies.

OBJECTIVE

To investigate the relationship between breast-feeding and obesity in childhood.

METHODS

Systematic review and meta-analysis of published epidemiological studies (cohort, case-control or cross-sectional studies) comparing early feeding-mode and adjusting for potential confounding factors. Electronic databases were searched and reference lists of relevant articles were checked. Calculations of pooled estimates were conducted in fixed- and random-effects models. Heterogeneity was tested by Q-test. Publication bias was assessed from funnel plots and by a linear regression method.

METHODS

Odds ratio (OR) for obesity in childhood defined as body mass index (BMI) percentiles.

RESULTS

Nine studies with more than 69,000 participants met the inclusion criteria. The meta-analysis showed that breast-feeding reduced the risk of obesity in childhood significantly. The adjusted odds ratio was 0.78, 95% CI (0.71, 0.85) in the fixed model. The assumption of homogeneity of results of the included studies could not be refuted (Q-test for heterogeneity, P>0.3), stratified analyses showed no differences regarding different study types, age groups, definition of breast-feeding or obesity and number of confounding factors adjusted for. A dose-dependent effect of breast-feeding duration on the prevalence of obesity was reported in four studies. Funnel plot regression gave no indication of publication bias.

CONCLUSIONS

Breast-feeding seems to have a small but consistent protective effect against obesity in children.

Neural stem cells with the characteristics of astrocytes persist in the subventricular zone (SVZ) of the juvenile and adult brain. These cells generate large numbers of new neurons that migrate through the rostral migratory stream to the olfactory bulb. The developmental origin of adult neural stem cells is not known. Here, we describe a lox-Cre-based technique to specifically and permanently label a restricted population of striatal radial glia in newborn mice. Within the first few days after labeling, these radial glial cells gave rise to neurons, oligodendrocytes, and astrocytes, including astrocytes in the SVZ. Remarkably, the rostral migratory stream contained labeled migratory neuroblasts at all ages examined, including 150-day-old mice. Labeling dividing cells with the S-phase marker BrdUrd showed that new neurons continue to be produced in the adult by precursors ultimately derived from radial glia. Furthermore, both radial glia in neonates and radial glia-derived cells in the adult lateral ventricular wall generated self-renewing, multipotent neurospheres. These results demonstrate that radial glial cells not only serve as progenitors for many neurons and glial cells soon after birth but also give rise to adult SVZ stem cells that continue to produce neurons throughout adult life. This study identifies and provides a method to genetically modify the lineage that links neonatal and adult neural stem cells.

The emerging notion of environment-induced reprogramming of Foxp3(+) regulatory T (Treg) cells into helper T (Th) cells remains controversial. By genetic fate mapping or adoptive transfers, we have identified a minor population of nonregulatory Foxp3(+) T cells exhibiting promiscuous and transient Foxp3 expression, which gave rise to Foxp3(-) ("exFoxp3") Th cells and selectively accumulated in inflammatory cytokine milieus or in lymphopenic environments including those in early ontogeny. In contrast, Treg cells did not undergo reprogramming under those conditions irrespective of their thymic or peripheral origins. Moreover, although a few Treg cells transiently lose Foxp3 expression, such "latent" Treg cells retained their memory and robustly re-expressed Foxp3 and suppressive function upon activation. This study establishes that Treg cells constitute a stable cell lineage, whose committed state in a changing environment is ensured by DNA demethylation of the Foxp3 locus irrespectively of ongoing Foxp3 expression.

CD4(+) regulatory T cells (T(reg) cells) that produce interleukin 10 (IL-10) are important contributors to immune homeostasis. We generated mice with a 'dual-reporter' system of the genes encoding IL-10 and the transcription factor Foxp3 to track T(reg) subsets based on coordinate or differential expression of these genes. Secondary lymphoid tissues, lung and liver had enrichment of Foxp3(+)IL-10(-) T(reg) cells, whereas the large and small intestine had enrichment of Foxp3(+)IL-10(+) and Foxp3(-)IL-10(+) T(reg) cells, respectively. Although negative for Il10 expression, both Foxp3(+) and Foxp3(-) CD4(+) thymic precursor cells gave rise to peripheral IL-10(+) T(reg) cells, with only Foxp3(-) precursor cells giving rise to all T(reg) subsets. Each T(reg) subset developed in IL-10-deficient mice, but this was blocked by treatment with antibody to transforming growth factor-beta. Thus, Foxp3(+) and Foxp3(-) precursor cells give rise to peripheral IL-10-expressing T(reg) cells by a mechanism dependent on transforming growth factor-beta and independent of IL-10.

The appearance of photosynthetic eukaryotes (algae and plants) dramatically altered the Earth's ecosystem, making possible all vertebrate life on land, including humans. Dating algal origin is, however, frustrated by a meager fossil record. We generated a plastid multi-gene phylogeny with Bayesian inference and then used maximum likelihood molecular clock methods to estimate algal divergence times. The plastid tree was used as a surrogate for algal host evolution because of recent phylogenetic evidence supporting the vertical ancestry of the plastid in the red, green, and glaucophyte algae. Nodes in the plastid tree were constrained with six reliable fossil dates and a maximum age of 3,500 MYA based on the earliest known eubacterial fossil. Our analyses support an ancient (late Paleoproterozoic) origin of photosynthetic eukaryotes with the primary endosymbiosis that gave rise to the first alga having occurred after the split of the Plantae (i.e., red, green, and glaucophyte algae plus land plants) from the opisthokonts sometime before 1,558 MYA. The split of the red and green algae is calculated to have occurred about 1,500 MYA, and the putative single red algal secondary endosymbiosis that gave rise to the plastid in the cryptophyte, haptophyte, and stramenopile algae (chromists) occurred about 1,300 MYA. These dates, which are consistent with fossil evidence for putative marine algae (i.e., acritarchs) from the early Mesoproterozoic (1,500 MYA) and with a major eukaryotic diversification in the very late Mesoproterozoic and Neoproterozoic, provide a molecular timeline for understanding algal evolution.