Cylindrospermopsin toxicity in mice following a 90-d oral exposure.

Cylindrospermopsin (CYN) is a toxin associated with numerous species of freshwater cyanobacteria throughout the world. It is postulated to have caused an episode of serious illnesses in Australia through treated drinking water, as well as lethal effects in livestock exposed to water from farm ponds. Toxicity included effects indicative of both hepatic and renal dysfunction. In humans, symptoms progressed from initial hepatomegaly, vomiting, and malaise to acidosis and hypokalemia, bloody diarrhea, and hyperemia in mucous membranes. Laboratory animal studies predominantly involved the intraperitoneal (i.p.) route of administration and confirmed this pattern of toxicity with changes in liver enzyme activities and histopathology consistent with hepatic injury and adverse renal effects. The aim of this study was designed to assess subchronic oral exposure (90 d) of purified CYN from 75 to 300 µg/kg/d in mouse. At the end of the dosing period, examinations of animals noted (1) elevated organ to body weight ratios of liver and kidney at all dose levels, (2) treatment-related increases in serum alanine aminotransferase (ALT) activity, (3) decreased blood urea nitrogen (BUN) and cholesterol concentrations in males, and (4) elevated monocyte counts in both genders. Histopathological alterations included hepatocellular hypertrophy and cord disruption in the liver, as well as renal cellular hypertrophy, tubule dilation, and cortical tubule lesions that were more prominent in males. A series of genes were differentially expressed including Bax (apoptosis), Rpl6 (tissue regeneration), Fabp4 (fatty acid metabolism), and Proc (blood coagulation). Males were more sensitive to many renal end points suggestive of toxicity. At the end of exposure, toxicity was noted at all dose levels, and the 75 µg/kg group exhibited significant effects in liver and kidney/body weight ratios, reduced BUN, increased serum monocytes, and multiple signs of histopathology indicating that a no-observed-adverse-effect level could not be determined for any dose level.

Canine mesenchymal stem cells are neurotrophic and angiogenic: An in vitro assessment of their paracrine activity.

Mesenchymal stem cells (MSCs) have been used in cell replacement therapies for connective tissue damage, but also can stimulate wound healing through paracrine activity. In order to further understand the potential use of MSCs to treat dogs with neurological disorders, this study examined the paracrine action of adipose-derived canine MSCs on neuronal and endothelial cell models. The culture-expanded MSCs exhibited a MSC phenotype according to plastic adherence, cell morphology, CD profiling and differentiation potential along mesenchymal lineages. Treating the SH-SY5Y neuronal cell line with serum-free MSC culture-conditioned medium (MSC CM) significantly increased SH-SY5Y cell proliferation (P <0.01), neurite outgrowth (P = 0.0055) and immunopositivity for the neuronal marker ßIII-tubulin (P = 0.0002). Treatment of the EA.hy926 endothelial cell line with MSC CM significantly increased the rate of wound closure in endothelial cell scratch wound assays (P = 0.0409), which was associated with significantly increased endothelial cell proliferation (P <0.05) and migration (P = 0.0001). Furthermore, canine MSC CM induced endothelial tubule formation in EA.hy926 cells in a soluble basement membrane matrix. Hence, this study has demonstrated that adipose-derived canine MSC CM stimulated neuronal and endothelial cells probably through the paracrine activity of MSC-secreted factors. This supports the use of canine MSC transplants or their secreted products in the clinical treatment of dogs with neurological disorders and provides some insight into possible mechanisms of action.

Wind observations above an urban river using a new lidar technique, scintillometry and anemometry.

Airflow along rivers might provide a key mechanism for ventilation in cities: important for air quality and thermal comfort. Airflow varies in space and time in the vicinity of rivers. Consequently, there is limited utility in point measurements. Ground-based remote sensing offers the opportunity to study 3D airflow in locations which are difficult to observe with conventional approaches. For three months in the winter and spring of 2011, the airflow above the River Thames in central London was observed using a scanning Doppler lidar, a scintillometer and sonic anemometers. First, an inter-comparison showed that lidar-derived mean wind-speed estimates compare almost as well to sonic anemometers (root-mean-square error (rmse) 0.65-0.68 ms(-1)) as comparisons between sonic anemometers (0.35-0.73 ms(-1)). Second, the lidar duo-beam operating strategy provided horizontal transects of wind vectors (comparison with scintillometer rmse 1.12-1.63 ms(-1)) which revealed mean and turbulent airflow across the river and surrounds; in particular, channelled airflow along the river and changes in turbulence quantities consistent with the roughness changes between built and river environments. The results have important consequences for air quality and dispersion around urban rivers, especially given that many cities have high traffic rates on roads located on riverbanks.

Toxicity and recovery in the pregnant mouse after gestational exposure to the cyanobacterial toxin, cylindrospermopsin.

Cylindrospermopsin (CYN) is a tricyclic alkaloid toxin produced by fresh water cyanobacterial species worldwide. CYN has been responsible for both livestock and human poisoning after oral exposure. This study investigated the toxicity of CYN to pregnant mice exposed during different segments of gestation. The course of recovery and individual responses to the toxin were evaluated. Adverse effects of CYN were monitored up to 7 weeks post-dosing by clinical examination, histopathology, biochemistry and gene expression. Exposure on gestational days (GD) 8-12 induced significantly more lethality than GD13-17 exposure. Periorbital, gastrointestinal and distal tail hemorrhages were seen in both groups. Serum markers indicative of hepatic injury (alanine amino transferase, aspartate amino transferase and sorbitol dehydrogenase) were increased in both groups; markers of renal dysfunction (blood urea nitrogen and creatinine) were elevated in the GD8-12 animals. Histopathology was observed in the liver (centrilobular necrosis) and kidney (interstitial inflammation) in groups exhibiting abnormal serum markers. The expression profiles of genes involved in ribosomal biogenesis, xenobiotic and lipid metabolism, inflammatory response and oxidative stress were altered 24 h after the final dose. One week after dosing, gross, histological and serum parameters had returned to normal, although increased liver/body weight ratio and one instance of gastrointestinal bleeding was found in the GD13-17 group. Gene expression changes persisted up to 2 weeks post-dosing and returned to normal by 4 weeks. Responses of individual animals to CYN exposure indicated highly significant inter-animal variability within the treated groups.

Flight periodicity and the vertical distribution of high-altitude moth migration over southern Britain.

The continuous operation of insect-monitoring radars in the UK has permitted, for the first time, the characterization of various phenomena associated with high-altitude migration of large insects over this part of northern Europe. Previous studies have taken a case-study approach, concentrating on a small number of nights of particular interest. Here, combining data from two radars, and from an extensive suction- and light-trapping network, we have undertaken a more systematic, longer-term study of diel flight periodicity and vertical distribution of macro-insects in the atmosphere. Firstly, we identify general features of insect abundance and stratification, occurring during the 24-hour cycle, which emerge from four years' aggregated radar data for the summer months in southern Britain. These features include mass emigrations at dusk and, to a lesser extent, at dawn and daytime concentrations associated with thermal convection. We then focus our attention on the well-defined layers of large nocturnal migrants that form in the early evening, usually at heights of 200-500 m above ground. We present evidence from both radar and trap data that these nocturnal layers are composed mainly of noctuid moths, with species such as Noctua pronuba, Autographa gamma, Agrotis exclamationis, A. segetum, Xestia c-nigrum and Phlogophora meticulosa predominating.

Activation-induced cellular accumulation of histamine in immature but not mature murine mast cells.

Mast cell activation involves the rapid release of inflammatory mediators, including histamine, from intracellular granules. The cells are capable of regranulation and multiple rounds of activation. The goal of this study was to determine if there are changes in the content of pre-formed mast cell mediators after a round of activation. After 24 h, the histamine content of bone marrow-derived mast cells (BMMC), but not that of peritoneal mast cells, exceeded the amount in resting cells. Accumulation of histamine in BMMC peaked at 72 h of activation, and returned toward preactivation levels by 96 h. The increase in histamine content was accompanied by an increase in the gene expression of histidine decarboxylase. No increases in beta hexosaminidase or murine mast cell protease-6 were observed. These findings indicate that BMMC respond to activation by increasing total cell-associated histamine content. This increase may be important to the response of these cells upon subsequent exposure to antigens.

Radar studies of the vertical distribution of insects migrating over southern Britain: the influence of temperature inversions on nocturnal layer concentrations.

Insects migrating over two sites in southern UK (Malvern in Worcestershire, and Harpenden in Hertfordshire) have been monitored continuously with nutating vertical-looking radars (VLRs) equipped with powerful control and analysis software. These observations make possible, for the first time, a systematic investigation of the vertical distribution of insect aerial density in the atmosphere, over temporal scales ranging from the short (instantaneous vertical profiles updated every 15 min) to the very long (profiles aggregated over whole seasons or even years). In the present paper, an outline is given of some general features of insect stratification as revealed by the radars, followed by a description of occasions during warm nights in the summer months when intense insect layers developed. Some of these nocturnal layers were due to the insects flying preferentially at the top of strong surface temperature inversions, and in other cases, layering was associated with higher-altitude temperature maxima, such as those due to subsidence inversions. The layers were formed from insects of a great variety of sizes, but peaks in the mass distributions pointed to a preponderance of medium-sized noctuid moths on certain occasions.

PD-L2 is a second ligand for PD-1 and inhibits T cell activation.

Programmed death I (PD-I)-deficient mice develop a variety of autoimmune-like diseases, which suggests that this immunoinhibitory receptor plays an important role in tolerance. We identify here PD-1 ligand 2 (PD-L2) as a second ligand for PD-1 and compare the function and expression of PD-L1 and PD-L2. Engagement of PD-1 by PD-L2 dramatically inhibits T cell receptor (TCR)-mediated proliferation and cytokine production by CD4+ T cells. At low antigen concentrations, PD-L2-PD-1 interactions inhibit strong B7-CD28 signals. In contrast, at high antigen concentrations, PD-L2-PD-1 interactions reduce cytokine production but do not inhibit T cell proliferation. PD-L-PD-1 interactions lead to cell cycle arrest in G0/G1 but do not increase cell death. In addition, ligation of PD-1 + TCR leads to rapid phosphorylation of SHP-2, as compared to TCR ligation alone. PD-L expression was up-regulated on antigen-presenting cells by interferon gamma treatment and was also present on some normal tissues and tumor cell lines. Taken together, these studies show overlapping functions of PD-L1 and PD-L2 and indicate a key role for the PD-L-PD-1 pathway in regulatingT cell responses.

Engagement of the PD-1 immunoinhibitory receptor by a novel B7 family member leads to negative regulation of lymphocyte activation.

PD-1 is an immunoinhibitory receptor expressed by activated T cells, B cells, and myeloid cells. Mice deficient in PD-1 exhibit a breakdown of peripheral tolerance and demonstrate multiple autoimmune features. We report here that the ligand of PD-1 (PD-L1) is a member of the B7 gene family. Engagement of PD-1 by PD-L1 leads to the inhibition of T cell receptor-mediated lymphocyte proliferation and cytokine secretion. In addition, PD-1 signaling can inhibit at least suboptimal levels of CD28-mediated costimulation. PD-L1 is expressed by antigen-presenting cells, including human peripheral blood monocytes stimulated with interferon gamma, and activated human and murine dendritic cells. In addition, PD-L1 is expressed in nonlymphoid tissues such as heart and lung. The relative levels of inhibitory PD-L1 and costimulatory B7-1/B7-2 signals on antigen-presenting cells may determine the extent of T cell activation and consequently the threshold between tolerance and autoimmunity. PD-L1 expression on nonlymphoid tissues and its potential interaction with PD-1 may subsequently determine the extent of immune responses at sites of inflammation.

Glucocorticoid receptor regulation in the rat embryo: a potential site for developmental toxicity?

Glucocorticoids play a key role in controlling numerous cellular processes during embryogenesis and fetal development. Excess glucocorticoids during development have been linked to dysmorphogenesis and/or intrauterine growth impairment in rodents. The actions of glucocorticoids are mediated by interaction with their receptors. Negative feedback regulation of glucocorticoid receptor (GR) is important for limiting cellular sensitivity to the hormones. Hence, acute exposure of the adult rat to the synthetic glucocorticoid dexamethasone (DEX) reduced both GR mRNA and protein in a variety of tissues that include hippocampus and liver, in a dose- and time-dependent fashion. Reduction in GR mRNA and protein were observable when DEX was given repeatedly at doses as low as 0. 05 mg/kg. In the control whole rat embryo, GR mRNA was low but measurable at as early as gestational day (GD) 10, but underwent rapid ontogenetic increase in the ensuring days. In contrast to the adult, neither GR mRNA nor protein in the whole rat embryo was affected by acute or repeated DEX administration to pregnant rats on GD10-13, even at doses as high as 0.8 mg/kg. Similar results were obtained in embryonic palate and liver, tissues known to be glucocorticoid targets. These data suggest that GR autoregulation does not occur during organogenesis in the rat. Accordingly, hormonal elevations from stress or chemical insults can be transduced unrestrictedly, ultimately leading to aberrant cell function and development. The unique mode of GR regulation seen in the embryonic cells may provide a potential common mechanism for developmental perturbation and toxicity for a variety of insults.

Identification of a human follicular dendritic cell molecule that stimulates germinal center B cell growth.

The initial interaction between B cells and follicular dendritic cells (FDCs) appears to be essential for germinal center (GC) formation. To identify molecules regulating this interaction, we generated FDC-staining monoclonal antibodies (mAbs) and screened them for their ability to block FDC-mediated costimulation of growth and differentiation of CD40-stimulated B cells. Using one of the inhibitory mAbs, 8D6, we expression cloned the cDNA encoding the 8D6 antigen (Ag) from a human FDC line, HK. The 8D6 Ag is a novel protein of 282 amino acids that is expressed abundantly on FDCs. Monolayers of COS cells transiently transfected with the 8D6 Ag cDNA stimulate B cell growth. The mAb 8D6 blocks the costimulatory function completely. The inhibitory activity of the mAb 8D6 was demonstrated to be due to an inhibition of cell cycle progression of CD40 ligand-stimulated GC B cells. In addition, the mAb 8D6 inhibits the growth of a lymphoma of GC origin, L3055, which depends on FDCs or HK cells for its growth. These findings suggest that the primary function of FDCs in the GC is to stimulate B cell growth. An FDC signal molecule, 8D6 Ag, may be an important molecule to mediate this function.

Adverse reproductive outcomes in the transgenic Ah receptor-deficient mouse.

The aryl hydrocarbon receptor (AHR) is a transcriptional regulatory protein that binds to upstream DNA response elements of target genes. Activation of the AHR by binding of ligands such as polyhalogenated dioxins, furans, and PCBs is associated with a wide range of adverse biological outcomes, including cancer, immune deficiencies, embryo/fetotoxicity, and reproductive toxicity. Investigations of the diverse biological responses mediated by the AHR led to production of a transgenic mouse in which the gene coding for the AhR was inactivated. AHR-deficient mice were fertile and at maturity exhibited immune system impairment and hepatic fibrosis. Our laboratory received several of these homozygous knockout (-/-) mice and mated them with wild-type (+/+) C57BL/6N mice to generate large numbers of heterozygotes (+/-). The -/- males were then mated with a total of 45 heterozygous +/- females. Offspring of these matings were genotyped and mated in all genotypic combinations. Although male and female -/- adults were fertile, the -/- females had difficulty maintaining conceptuses during pregnancy, surviving pregnancy and lactation, and rearing pups to weaning. Only 46% of the 39 pregnant -/- females successfully raised pups to weaning. The -/- pups showed poor survival during lactation (average death rate per litter was 16%) and after weaning (26.5% of the 230 weaned -/- pups died within 2 weeks). Only 39% of the implantations in uteri of -/- dams resulted in offspring surviving to Postnatal Day 45. Across all litters the sex ratios and genotypic frequencies were comparable to expected values. Reproductive success was adversely affected in Ahr-null females and conceptuses. Additional study is needed to reveal the etiology of these effects.

AhR, ARNT, and CYP1A1 mRNA quantitation in cultured human embryonic palates exposed to TCDD and comparison with mouse palate in vivo and in culture.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is developmentally toxic in many species and induces cleft palate in the C57BL/6N mouse embryo. Palatogenesis in mouse and human embryos involves homologous processes at the morphological, cellular, and molecular levels. In organ culture, mouse and human palates respond similarly to TCDD. The present study quantitates the expression of AhR, ARNT, and CYP1A1 mRNA in human embryonic palates in organ culture. Palatal tissues were exposed to 1 x 10(-10), 1 x 10(-9), or 1 x 10(-8) M TCDD or control medium and sampled at 0, 2, 4, and 6 hours for quantitative RT-PCR using a synthetic RNA internal standard. Similar measurements of CYP1A1 gene expression were collected for mouse palates cultured in this model. In human palates, AhR expression correlated with ARNT and CYP1A1 mRNA expression. TCDD induction of CYP1A1 was time- and concentration-dependent. The expression of these genes presented a uniform and continuous distribution across the group of embryos, with no subset of either high or low expressors/responders. The ratio of AhR to ARNT was approximately 4:1. AhR mRNA increased during the culture period in both treated and control subjects; however, ARNT expression was relatively constant. TCDD did not alter either AhR or ARNT expression in a consistent dose- or time-related manner. Comparison of human and mouse data showed a high correlation across species for the induction of CYP1A1. Human embryos expressed approximately 350 times less AhR mRNA than the mouse, and in earlier studies it was shown that human palates required 200 times more TCDD to produce the same effects. When the morphological, cellular, and molecular responses to TCDD between mouse and human are compared, it seems highly unlikely that human embryos could be exposed to sufficient TCDD to achieve changes in palatal differentiation that would lead to cleft palate.

RT-PCR quantification of AHR, ARNT, GR, and CYP1A1 mRNA in craniofacial tissues of embryonic mice exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin and hydrocortisone.

C57BL/6N mouse embryos exposed to hydrocortisone (HC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) develop cleft palate. An interaction between these agents produces clefts at doses which alone are not teratogenic. The glucocorticoid receptor (GR) and dioxin receptor (AhR) mediated these responses and their gene expression was altered by TCDD and/or HC in palates examined on gestation day (GD) 14 by Northern blot analysis and in situ hybridization. The present study quantifies AhR, AhR nuclear translocator (ARNT), and GR mRNA at 4, 12, 24, and 48 h after exposure (time 0 = dose administration at 8 A.M. on gestation day 12) on GD12 to TCDD (24 micrograms/kg), HC (100 mg/kg) or HC (25 mg/kg) + TCDD (3 micrograms/kg). The induction of CYP1A1 mRNA was also quantified at 2, 4, 6, 12, 24, and 48 h for control and TCDD-exposed samples. Total RNA was prepared from midfacial tissue of 4-6 embryos/litter at each time and dose. An RNA internal standard (IS) for each gene was synthesized, which included the gene's primer sequences separated by a pUC19 plasmid sequence. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on total RNA + IS using a range of 5-7 IS concentrations across a constant level of total RNA. PCR products were separated in gels (mRNA and IS-amplified sequences differed by 30-50 bases), ethidium bromide-stained, imaged (Hamamatsu Photonics Systems, Bridgewater, NJ), and quantified with NIH Image. CYP1A1 mRNA was significantly induced in the TCDD-exposed samples at all time points examined (p = 0.005 at 2 h and 0.001 after 2 h). During palatal shelf outgrowth on GD12, AhR mRNA levels increased significantly and this was not affected by treatment with TCDD or HC + TCDD. A significant increase in GR was detected at 24 h (p < 0.05) and this was unaffected by any of the exposures. Expression of ARNT increased at 12 h (p < 0.001); however, treatment with HC or HC + TCDD blocked this increase (p < 0.05). At 24 h, the TCDD-treated embryos had significantly lower ARNT mRNA compared with controls (p < 0.001). The relative overall expression level of the genes was AhR > ARNT > GR. Within individuals, expression of AhR and/or ARNT was highly correlated with GR level. In conclusion, CYP1A1 mRNA was expressed in developing craniofacial tissue and was highly induced by TCDD exposure. AhR, ARNT, and GR mRNA are upregulated in early palatogenesis, although not on the same schedule. The TCDD-induced decrease in ARNT at 24 h after dosing and the HC and HC + TCDD-induced delay in upregulation of ARNT may affect the dynamics of heterodimer formation between AhR and ARNT. The changes in ARNT mRNA level could also affect availability of this transcriptional regulator to interact with other potential partners, and these effects, separately or in combination, may be involved in disruption of normal embryonic development.

The murine IL-13 receptor alpha 2: molecular cloning, characterization, and comparison with murine IL-13 receptor alpha 1.

Two components of a receptor complex for IL-13, the IL-4R and a low affinity IL-13-binding chain, IL-13R alpha 1, have been cloned in mice and humans. An additional high affinity binding chain for IL-13, IL-13R alpha 2, has been described in humans. We isolated a cDNA from the thymus that encodes the murine orthologue of the human IL-13R alpha 2. The predicted protein sequence of murine IL-13R alpha 2 (mIL-13R alpha 2) has 59% overall identity to human IL-13R alpha 2 and is closely related to the murine low affinity IL-13-binding subunit, IL-13R alpha 1. The genes for both mIL-13-binding chains map to the X chromosome. A specific interaction between mIL-13R alpha 2.Fc protein and IL-13 was demonstrated by surface plasmon resonance using a BIACORE instrument. Ba/F3 cells that were transfected with mIL-13R alpha 2 expressed 5000 molecules per cell and bound IL-13 with a single Kd of 0.5 to 1.2 nM. However, these cells did not proliferate in response to IL-13, and the IL-4 dose response was unaffected by high concentrations of IL-13. In contrast, the expression of mIL-13R alpha 1 by Ba/F3 cells resulted in a sensitive proliferative response to IL-13. Consistent with its lower affinity for IL-13, IL-13R alpha 1.Fc was 100-fold less effective than IL-13R alpha 2.Fc in neutralizing IL-13 in vitro. These results show that mIL-13R alpha 2 and mIL-13R alpha 1 are not functionally equivalent and predict distinct roles for each polypeptide in IL-13R complex formation and in the modulation of IL-13 signal transduction.

Characterization of murine Flt4 ligand/VEGF-C.

Flt4 is a receptor protein tyrosine kinase that is expressed in the adult lymphatic endothelium and high endothelial venules. We have used a BIAcore assay to identify rodent and human cell conditioned media containing the ligand of Flt4 (Flt4-L). Receptor-based affinity chromatography was used to purify this growth factor, followed by amino acid sequencing and molecular cloning of the murine cDNA, the orthologue of human vascular endothelial growth factor-C and vascular endothelial growth factor related protein. The murine flt4-L gene was localized to chromosome 8 and demonstrated to be widely expressed. Flt4-L was found to have a hydrophobic signal sequence and a pro-peptide-like sequence that is removed to generate the mature N-terminus. In addition, the C-terminal region of Flt4-L has four repeats of a cysteine-rich motif that is presumably also proteolytically processed to generate the 21000 Mr polypeptide subunit of the Flt4-L homodimer. Recombinant Flt4-L activated Flt4 as judged by induction of tyrosyl phosphorylation, and induced mitogenesis in vitro of lymphatic endothelial cells.

Expression of murine interleukin 11 and its receptor alpha-chain in adult and embryonic tissues.

Interleukin 11 (IL-11) is a multifunctional cytokine that has diverse effects on blood cells and their precursors and on a number of cell types outside of the hematopoietic system. The cDNAs encoding murine IL-11 and its receptor alpha-chain (IL-11R alpha) have recently been isolated. We have used the RNase protection assay to examine the expression of murine IL-11 and IL-11R alpha in a range of adult mouse tissues, in embryos, and during development of embryonic stem (ES) cells into cystic embryoid bodies in vitro. The testis showed a high level of IL-11 gene expression while a much lower level of expression was detected in the lung, stomach, small intestine, and large intestine. Expression of IL-11 was not detected between day 10.5 and day 18.5 post coitum of embryonic development or in differentiating ES cells in vitro. In contrast, the IL-11R alpha was found to be expressed in all adult tissues examined, during embryonic development, and in totipotent and differentiating ES cells.

Molecular cloning and characterization of murine interleukin-11.

Human interleukin-11 (IL-11) has been shown to have pleiotropic action on hematopoietic, hepatic, stromal, epithelial, neural, and osteoclast cells. In the present work, the murine IL-11 cDNA has been isolated from a fetal thymic cell line, and its structure and function compared with human IL-11. The murine protein was demonstrated to have identical actions on the proliferation of a murine plasmacytoma cell line, murine primitive bone marrow progenitor cells, and megakaryocyte precursors. The murine IL-11 protein was synthesized as a soluble thioredoxin-IL-11 fusion in Escherichia coli and the expression of murine IL-11 was examined by pulse-chase radiolabeling in COS cells. The chromosomal location of the murine IL-11 gene was assigned to the proximal arm of chromosome 7.

Regulation of flt-1 expression during mouse embryogenesis suggests a role in the establishment of vascular endothelium.

Flt-1 is a high affinity binding receptor for the vascular endothelial cell growth factor (VEGF) and is primarily expressed in endothelial cells. In this study we have investigated the temporal and spatial regulation of its expression by establishing mouse lines containing the lacZ gene targeted into the flt-1 locus through homologous recombination in embryonic stem (ES) cells. In the yolk sac as well as in the embryo proper, lacZ expression faithfully reflected the endogenous expression pattern of the flt-1 gene. LacZ staining of heterozygous embryos led to the following observations: (1) the onset of flt-1 expression is detected at the early primitive streak stage in the extraembryonic mesoderm, and is strongly up-regulated thereafter, reaching a maximum by early to midsomite stages and declining subsequently; (2) while flt-1 is widely expressed within the developing vascular endothelium, its expression level is differentially regulated both spatially and temporally. The pattern of flt-1 expression suggests that it may play an important role in the initiation of endothelium development; and (3) flt-1 is expressed in essentially all the cells in early blood islands, but later its expression is gradually restricted to the endothelial lineage. Our results indicate that flt-1 is a marker for hemangioblasts, the presumed progenitor for both hematopoietic and angioblastic lineage. The flt-1 expression pattern also suggests that it may play important roles in both vasculogenesis and angiogenesis.

A role for CD9 molecules in T cell activation.

Costimulation mediated by the CD28 molecule plays an important role in optimal activation of T cells. However, CD28-deficient mice can mount effective T cell-dependent immune responses, suggesting the existence of other costimulatory systems. In a search for other costimulatory molecules on T cells, we have developed a monoclonal antibody (mAb) that can costimulate T cells in the absence of antigen-presenting cells (APC). The molecule recognized by this mAb, 9D3, was found to be expressed on almost all mature T cells and to be a protein of approximately 24 kD molecular mass. By expression cloning, this molecule was identified as CD9, 9D3 (anti-CD9) synergized with suboptimal doses of anti-CD3 mAb in inducing proliferation by virgin T cells. Costimulation was induced by independent ligation of CD3 and CD9, suggesting that colocalization of these two molecules is not required for T cell activation. The costimulation by anti-CD9 was as potent as that by anti-CD28. Moreover, anti-CD9 costimulated in a CD28-independent way because anti-CD9 equally costimulated T cells from the CD28-deficient as well as wild-type mice. Thus, these results indicate that CD9 serves as a molecule on T cells that can deliver a potent CD28-independent costimulatory signal.