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Sexual selection via male competition is a strong evolutionary force that can drive rapid changes in competitive traits and subsequently lead to population divergence and speciation. Territorial males of many odonates are known to use their colorful wings as visual signals and to perform agonistic displays toward intruders. Psolodesmus mandarinus dorothea and Psolodesmus mandarinus mandarinus are two parapatrically distributed sister damselflies that share similar ecological characteristics but differ markedly in wing coloration. The wings of P. m. dorothea are mostly clear, whereas those of P. m. mandarinus have a large area of black pigmentation and a central white patch. We investigated whether territorial males of the two damselflies at breeding sites display distinct agonistic behaviors associated with their respective wing colors. Behavioral interactions between territorial and intruder males and their wing kinematics were filmed and analyzed for P. m. dorothea in Lienhuachih of central Taiwan, and P. m. mandarinus in Tianxiyuan and Fusan of northern Taiwan. We observed that the P. m. mandarinus males exhibited a novel set of perched wing displays, which was not only absent in its sister P. m. dorothea but also previously unknown in Odonata. At breeding sites, perched rival males of P. m. mandarinus with pigmented wings exhibited escalating agonistic wing-flapping and wing-hitting displays toward each other. In contrast, territorial males of P. m. dorothea with clear wings engaged only in aerial chase or face-to-face hovering when intruder males approached from the air. These results indicate that the two sister P. mandarinus damselflies diverged behaviorally in territorial contests and support the hypothesis of coadaptation on the basis of wing colors and types of wing movement in Odonata. Our findings further suggest that divergent agonistic wing displays may play a pivotal role in the speciation mechanism of P. mandarinus damselflies. The sequential analyses of behavioral characteristics and progression suggest that P. m. mandarinus damselflies likely use mutual assessment of rivals in territorial contests.

The development of new mechanisms of resistance among pathogens, the occurrence and transmission of genes responsible for antibiotic insensitivity, as well as cancer diseases have been a serious clinical problem around the world for over 50 years. Therefore, intense searching of new leading structures and active substances, which may be used as new drugs, especially against strain resistant to all available therapeutics, is very important. Dihydrofolate reductase (DHFR) has attracted a lot of attention as a molecular target for bacterial resistance over several decades, resulting in a number of useful agents. Trimethoprim (TMP), (2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine) is the well-known dihydrofolate reductase inhibitor and one of the standard antibiotics used in urinary tract infections (UTIs). This review highlights advances in design, synthesis, and biological evaluations in structural modifications of TMP as DHFR inhibitors. In addition, this report presents the differences in the active site of human and pathogen DHFR. Moreover, an excellent review of DHFR inhibition and their relevance to antimicrobial and parasitic chemotherapy was presented.

In humans and some other mammals and birds, the development of communication systems requires social feedback. How do such systems evolve from ancestral states featuring innate developmental mechanisms? We report evidence of a novel form of social ontogeny in the communication system of Enchenopa treehoppers that suggests an answer to this question. These insects use plant-borne vibrational signals throughout their lives. Signal repertoires of nymphs and adults differed and showed sexually dimorphic ontogenetic trajectories; individual differences projected into some of the features of adult signals and mate preferences. Signals and mate preferences differed between adults reared in isolation and adults reared in groups, but even individuals reared in isolation developed species-typical signals. In this type of social ontogeny, peer inputs cause variation in signals and preferences. Thus, even innate communication systems can be socially malleable. This may set the stage for the evolution of obligate social feedbacks in communication: the starting point is already socially plastic and does not require learning to arise de novo.

The aim of the study was to evaluation of the suitability of broiler chicken and turkey commercial feed mixes in the diets of grey partridge (Perdix perdix L.) during the rearing period. The study was carried out on a farm authorized by the Minister of the Environment to conduct breeding and farming of grey partridge, DL Pgł-671323/15852/13/RN dated 24 April 2013. The research material consisted of grey partridge chicks assigned to two groups: one, fed the complete feed mix for turkeys IB-1 till 4 weeks of rearing, followed by the complete feed mix for turkeys B-2 from 5 weeks on (group I, n = 22) and the other, fed the complete feed DKA-Starter for broiler chickens (group II, n = 22). The rearing cycle took 8 weeks. Observations within the experiment included deaths records and monitoring of health status, body weight, feed intake and maintenance conditions. Better parameters of partridges rearing were attained as a result of IB feeding. Birds fed with IB-1 and IB-2 feed mixes showed a higher body weight, as compared to chicks fed DKA-Starter (DKA-S). The difference persisted from the first week of rearing to the end of the feeding trial (respectively 0.32 g and 4.59 g). From day 1 until 8 weeks of age, partridges fed the IB feed showed greater average daily gains (4.17 g), a faster rate of growth (181.67%) and a greater final body weight, all at a lower feed intake compared to those fed mix with DKA-S. It has been found that the IB feed mix can successfully be used in the nutrition of grey partridges., Celem pracy była ocena przydatności mieszanek paszowych dla kurcząt brojlerów i indyków w dietach kuropatwy szarej (Perdix perdix L.) w okresie odchowu. Badania przeprowadzono na fermie, posiadającej zgodę Ministra Środowiska na prowadzenie chowu i hodowli zamkniętej kuropatwy zwyczajnej DL Pgł-6713-23/15852/13/RN z dnia 24.04.2013 r. Materiał badawczy stanowiły kurczęta kuropatwy. Kurczęta kuropatwy zwyczajnej podzielono na dwie grupy: żywione mieszanką paszową pełnoporcjową dla indyków IB-1 do 4. tygodnia odchowu i mieszanką paszową pełnoporcjową dla indyków IB-2 od 5 tygodnia odchowu (grupa I - 22 sztuki) oraz żywione mieszanką paszową pełnoporcjową dla kurcząt brojlerów DKA-Starter (grupa II - 22 sztuki). Okres odchowu trwał 8 tygodni. W czasie realizacji badań kontrolowano i rejestrowano upadki, stan zdrowotny, masę ciała ptaków, zużycie paszy i warunki utrzymania. Lepsze wyniki odchowu kuropatwy uzyskano w wyniku zastosowania mieszanki paszowej IB. W grupie ptaków żywionych mieszanką paszową IB-1 i IB-2 wykazano większą masę ciała ptaków, w porównaniu do piskląt żywionych mieszanką paszową DKA-Starter (DKA-S). Różnica utrzymywała się od pierwszego tygodnia odchowu do końca okresu doświadczalnego (odpowiednio: 0,32 g i 4,59 g). U kuropatw żywionych mieszanką paszową IB obserwowano od 1. dnia do 8. tygodnia życia większe średnie dobowe przyrosty (4,17 g), większe tempo wzrostu (181,67%) oraz większą, końcową masę ciała przy mniejszym spożyciu paszy niż u kuropatw żywionych mieszanką paszową DKA-S. Stwierdzono, że mieszanka paszowa IB może być z powodzeniem stosowana, jako pasza w żywieniu kuropatwy zwyczajnej.

Social causes of variation in animal communication systems have important evolutionary consequences, including speciation. The relevance of these effects depends on how widespread they are among animals. There is evidence for such effects not only in birds and mammals, but also frogs and some insects and spiders. Here we analyse the social ontogeny of adult mate preferences in an insect, Enchenopa treehoppers. In these communal plant-feeding insects, individuals reared in isolation or in groups differ in their mate preferences, and the group-reared phenotype can be rescued by playbacks to isolation-reared individuals. We ask about the relative role of signalling experience and signalling practice during ontogeny on the development of adult mating preferences in Enchenopa females. Taking advantage of variation in the signal experience and signalling practice of isolation-reared individuals, we find switch-like effects for experience and practice on female mate preference phenotypes, with individuals having some experience and practice as juveniles best rescuing the group-reared preference phenotype. We discuss how understanding the nature and distribution of social-ontogenetic causes of variation in mate preferences and other sexual traits will bring new insights into how within- and between-population variation influences the evolution of communication systems. This article is protected by copyright. All rights reserved.

A new series of trimethoprim (TMP) analogs containing amide bonds (1&ndash, 6) have been synthesized. Molecular docking, as well as dihydrofolate reductase (DHFR) inhibition assay were used to confirm their affinity to bind dihydrofolate reductase enzyme. Data from the ethidium displacement test showed their DNA-binding capacity. Tests confirming the possibility of DNA binding in a minor groove as well as determination of the association constants were performed using calf thymus DNA, T4 coliphage DNA, poly (dA-dT)2 and poly (dG-dC)2. Additionally, the mechanism of action of the new compounds was studied. In conclusion, some of our new analogs inhibited DHFR activity more strongly than TMP did, which confirms, that the addition of amide bonds into the analogs of TMP increases their affinity towards DHFR.

Social plasticity may be an important originator of divergence in mating signals and other sexual traits. Understanding the evolutionary causes and consequences of social plasticity requires analyzing how different features of the social environment influence the expression of signals and preferences. Here we focus on experience of signaling environments. We adopt the vantage point of a hypothetical focal juvenile individual, and ask whether its experience of the interactions between other individuals in the group would vary across groups of different size and species composition. We worked with Enchenopa treehoppers, group-living herbivorous insects that communicate with plant-borne vibrational signals as juveniles and adults. We manipulated group composition and size experimentally and monitored the behavior of the juvenile treehoppers. We found that the treehoppers’ signaling rates varied with group type, size, and disturbance. Although our results likely underestimate the range of variation in behavio...

Background Hypoglycemia (HG) is common in IUGR neonates. In normally grown (NG) neonatal rats, acute HG causes neuronal injury in the brain, cerebral cortex more vulnerable than the hippocampus (HPC). We hypothesized that the IUGR brain is less vulnerable to hypoglycemia-induced injury while preserving the regional variation in vulnerability. Methods We induced IUGR via bilateral uterine artery ligation on gestational day 19 (term 22d) rats. On postnatal day 14, insulin-induced HG of equivalent severity and duration (blood glucose

article i nfo Article history: This paper estimates the value of water rights in a rapidly urbanizing semi-arid area: Phoenix, Arizona. To do this we use hedonic pricing to explore the impact of water rights on property values in 151 agricultural land transactions that occurred between 2001 and 2005. We test two main hypotheses: (1) that the marginal will- ingness to pay for water rights is higher in more developed urbanizing areas than in less developed rural areas, and (2) that the marginal willingness to pay for water rights in urban areas is increasing in the value of developed land. We find that the marginal willingness to pay for water rights is highest among properties in urbanized or urbanizing areas where a significant proportion of the land has already been developed. Additionally, we find that the marginal willingness to pay for agricultural water rights is greatest in cities where developed land is most valuable.

Amino acids and glucose acutely stimulate fetal insulin secretion. In isolated adult pancreatic islets, amino acids potentiate glucose-stimulated insulin secretion (GSIS), but whether amino acids have this same effect in the fetus is unknown. Therefore, we tested the effects of increased fetal amino acid supply on GSIS and morphology of the pancreas. We hypothesized that increasing fetal amino acid supply would potentiate GSIS. Singleton fetal sheep received a direct intravenous infusion of an amino acid mixture (AA) or saline (CON) for 10–14 days during late gestation to target a 25–50% increase in fetal branched-chain amino acids (BCAA). Early-phase GSIS increased 150% in the AA group ( P < 0.01), and this difference was sustained for the duration of the hyperglycemic clamp (105 min) ( P < 0.05). Glucose-potentiated arginine-stimulated insulin secretion (ASIS), pancreatic insulin content, and pancreatic glucagon content were similar between groups. β-Cell mass and area were unchanged between groups. Baseline and arginine-stimulated glucagon concentrations were increased in the AA group ( P < 0.05). Pancreatic α-cell mass and area were unchanged. Fetal and pancreatic weights were similar. We conclude that a sustained increase of amino acid supply to the normally growing late-gestation fetus potentiated fetal GSIS but did not affect the morphology or insulin content of the pancreas. We speculate that increased β-cell responsiveness (insulin secretion) following increased amino acid supply may be due to increased generation of secondary messengers in the β-cell. This may be enhanced by the paracrine action of glucagon on the β-cell.

Maternal high-protein supplements designed to increase birth weight have not been successful. We recently showed that maternal amino acid infusion into pregnant sheep resulted in competitive inhibition of amino acid transport across the placenta and did not increase fetal protein accretion rates. To bypass placental transport, singleton fetal sheep were intravenously infused with an amino acid mixture (AA, n = 8) or saline [control (Con), n = 10] for ∼12 days during late gestation. Fetal leucine oxidation rate increased in the AA group (3.1 ± 0.5 vs. 1.4 ± 0.6 μmol·min−1·kg−1, P < 0.05). Fetal protein accretion (2.6 ± 0.5 and 2.2 ± 0.6 μmol·min−1·kg−1in AA and Con, respectively), synthesis (6.2 ± 0.8 and 7.0 ± 0.9 μmol·min−1·kg−1in AA and Con, respectively), and degradation (3.6 ± 0.6 and 4.5 ± 1.0 μmol·min−1·kg−1in AA and Con, respectively) rates were similar between groups. Net fetal glucose uptake decreased in the AA group (2.8 ± 0.4 vs. 3.9 ± 0.1 mg·kg−1·min−1, P < 0.05). The glucose-O2quotient also decreased over time in the AA group ( P < 0.05). Fetal insulin and IGF-I concentrations did not change. Fetal glucagon increased in the AA group (119 ± 24 vs. 59 ± 9 pg/ml, P < 0.05), and norepinephrine (NE) also tended to increase in the AA group (785 ± 181 vs. 419 ± 76 pg/ml, P = 0.06). Net fetal glucose uptake rates were inversely proportional to fetal glucagon ( r2= 0.38, P < 0.05), cortisol ( r2= 0.31, P < 0.05), and NE ( r2= 0.59, P < 0.05) concentrations. Expressions of components in the mammalian target of rapamycin signaling pathway in fetal skeletal muscle were similar between groups. In summary, prolonged infusion of amino acids directly into normally growing fetal sheep increased leucine oxidation. Amino acid-stimulated increases in fetal glucagon, cortisol, and NE may contribute to a shift in substrate oxidation by the fetus from glucose to amino acids.

Background and purpose The aim of the study was to present our results of the surgical treatment of subtemporal fossa tumours and surrounding regions using the extended subtemporal approach. Material and methods Twenty-five patients (10 women, 15 men) with subtemporal fossa tumours were included in the study. The neurological and performance status of the patients were assessed before and after surgery as well as at the conclusion of treatment. The approximate volume of the operated tumour, its relation to large blood vessels and cranial nerves, as well as consistency and vascularisation were assessed. Results The symptom duration ranged from 2 to 80 months (mean: 14 months). In 44% of patients, headache was the predominant symptom. Less frequent symptoms were: paralysis of the abducent nerve and disturbances of the trigeminal nerve. Approximate volume of the tumours ranged from 13 to 169 cm 3 (mean: 66 cm 3 ). The most frequent histological diagnosis was meningioma (16%), followed by angiofibroma, neurinoma and adenocystic carcinoma (12%). Total or subtotal resection was achieved in 80% of patients. Conclusions The extended subtemporal approach allows for the removal of tumours of the subtemporal fossa and surrounding regions. This approach also allows one to remove tumours expanding in the regions surrounding the subtemporal fossa only. In such cases the subtemporal fossa constitutes the way of the surgical approach.

Summary miR-24, upregulated during terminal differentiation of multiple lineages, inhibits cell-cycle progression. Antagonizing miR-24 restores postmitotic cell proliferation and enhances fibroblast proliferation, whereas overexpressing miR-24 increases the G1 compartment. The 248 mRNAs downregulated upon miR-24 overexpression are highly enriched for DNA repair and cell-cycle regulatory genes that form a direct interaction network with prominent nodes at genes that enhance ( MYC , E2F2 , CCNB1 , and CDC2 ) or inhibit ( p27Kip1 and VHL ) cell-cycle progression. miR-24 directly regulates MYC and E2F2 and some genes that they transactivate. Enhanced proliferation from antagonizing miR-24 is abrogated by knocking down E2F2 , but not MYC , and cell proliferation, inhibited by miR-24 overexpression, is rescued by miR-24-insensitive E2F2 . Therefore, E2F2 is a critical miR-24 target. The E2F2 3′UTR lacks a predicted miR-24 recognition element. In fact, miR-24 regulates expression of E2F2 , MYC , AURKB , CCNA2 , CDC2 , CDK4 , and FEN1 by recognizing seedless but highly complementary sequences.

In the gram-positive pathogen Staphylococcus aureus, transposon mutagenesis is a useful method of screening large numbers of mutants for a given phenotype. However, constructing a transposon mutant library can take several months of work and validation in the laboratory. In this chapter, we describe a method for batch transduction of existing transposon mutations into new genetic backgrounds. Transduction in S. aureus is accomplished quickly and easily in most commonly used laboratory strains. The method described herein utilizes transduction to facilitate the rapid creation of new libraries and quick screening in strains containing phenotypic reporter constructs.

Viral heterogeneity is a major hurdle for potential therapeutic use of RNA interference (RNAi) against HIV-1. To determine the extent of RNAi tolerance to mutations, we tested 3 viral target sites with differing propensity for mutations: a highly variable rev sequence, a gag sequence conserved only among clade B isolates, and a vif sequence highly conserved across clades. Lentiviral expression of all 3 shRNAs inhibited replication of the homologous HIVIIIB strain. However, they differed in their ability to protect primary CD4 T cells against multiple isolates within and across HIV clades. The least conserved rev sequence inhibited only 2 of 5 clade B isolates. The gag sequence (conserved within clade B) protected 5 of 5 clade B isolates but not other clade viruses with 2 or 3 mutations in the central region. In contrast, the vif sequence, which was conserved across clades except for single mutations at positions 14 and 17, inhibited viruses from 5 different clades. Moreover, siRNAs with introduced mutations at sites of gag sequence polymorphisms showed reduced antiviral activity, whereas mutations in vif siRNA only modestly decreased silencing. Thus, although 1 or 2 mutations at peripheral sites are tolerated, mutations in the central target cleavage region abolish RNAi activity.

This article presents a method of analysis of variation in the cortical branching of the posterior cerebral artery (PCA). The method takes account of previous observations and reflects various symptoms of such variation. Of 100 cerebral hemispheres into whose vessels a contrast medium was injected, 94 hemispheres have been qualified for further examination. In six hemispheres, there were anomalies of the PCA, which are not included in the present analysis of division. A general model of the division of the artery has been developed. On the basis of this model, principles of the analysis of the variation have been discussed. The model comprises of three elements. The first is the main trunk. The second is intermediate trunks. The third element may appear in two forms: proper cortical arteries and cortical arterial groups. The main trunk was defined as the vessels arising from the final division of the basilar artery with its end in the place of branching of the most peripheral proper cortical artery, that is, parieto-occipital artery. Intermediate trunks are these parts of the PCA that extend between the main trunk and proper cortical arteries or cortical arterial groups. The identification criterion for the intermediate trunks is its length, which is to be equal to or exceed the sum of the radius of the two vessels, which the intermediate trunk gives off. There have been distinguished primary and secondary intermediate trunks. Proper cortical arteries or cortical arterial groups are parts of the PCA by means of which blood is supplied directly to the cortex. Cortical branches of the PCA of a relatively constant topography and vascularization were termed proper cortical arteries. In some cases, cortical branches are accompanied by small vessels of varying number and size. They were named accessory cortical arteries. Proper cortical arteries together with accompanying accessory cortical arteries have been named cortical arterial groups. The number of the proper cortical arteries is considered to be constant and is six. The theory of combinations has been applied to establish all possible branching variants. There are 16 such variants. Eleven of them (68%) were represented in the analyzed material. In seven hemispheres (7.4%), intermediate trunks were not observed. In 87 hemispheres (92.6%), there were present primary intermediate trunks. In four hemispheres (4.2%), there were found primary and secondary intermediate trunks. Both proper cortical arteries and cortical arterial groups reached the first five cortical areas of the PCA. The last cortical area was reached by a cortical arterial group only.

Previous work has established that CD24 is a costimulatory molecule for T-cell clonal expansion. Studies using CD24 -/- mice demonstrated that CD24 plays a critical role in the CD28-independent immune response against virus and soluble antigens. The role of CD24 on dendritic cells (DCs) has not been reported. Here, we compare the CD24(+/+) and CD24(-/-) DCs in the induction of initial clonal expansion and elicitation of memory CD4(+) T cells in vivo. Our results demonstrate that the CD24 expressed on DCs is essential neither for the induction of initial T-cell clonal expansion nor for elicitation of memory activity of primed T cells in vivo.

The adaptive immune system can generate distinct classes of responses, but the mechanisms that determine this are poorly understood. In this study, we demonstrate that different Toll-like receptor (TLR) ligands induce distinct dendritic cell (DC) activation and immune responses in vivo. Thus, Escherichia coli LPS (TLR-4 stimulus), activates DCs to produce abundant IL-12(p70), but little IL-10, and stimulates Th1 and Tc1 responses. In contrast, Pam-3-cys (TLR-2 stimulus) elicits less IL-12(p70), but abundant IL-10, and favors Th2 and T cytotoxic 2 (Tc2) responses. These distinct responses likely occur via differences in extracellular signal-regulated kinase signaling in DCs. Thus, Pam-3-cys induces enhanced extracellular signal-regulated kinase signaling, compared with LPS, resulting in suppressed IL-12(p70) and enhanced IL-10 production, as well as enhanced induction of the transcription factor, c-Fos. Interestingly, DCs from c-fos−/− mice produce more IL-12(p70), but less IL-10, compared with control DCs. Therefore, different TLR ligands induce distinct cytokines and signaling in DCs, and differentially bias Th responses in vivo.

Recent evidence suggests that in addition to their well known stimulatory properties, dendritic cells (DCs) may play a major role in peripheral tolerance. It is still unclear whether a distinct subtype or activation status of DC exists that promotes the differentiation of suppressor rather than effector T cells from naive precursors. In this work, we tested whether the naturally occurring CD4+ CD25+ regulatory T cells (Treg) may control immune responses induced by DCs in vivo. We characterized the immune response induced by adoptive transfer of antigen-pulsed mature DCs into mice depleted or not of CD25+ cells. We found that the development of major histocompatibility complex class I and II–restricted interferon γ–producing cells was consistently enhanced in the absence of Treg. By contrast, T helper cell (Th)2 priming was down-regulated in the same conditions. This regulation was independent of interleukin 10 production by DCs. Of note, splenic DCs incubated in vitro with Toll-like receptor ligands (lipopolysaccharide or CpG) activated immune responses that remained sensitive to Treg function. Our data further show that mature DCs induced higher cytotoxic activity in CD25-depleted recipients as compared with untreated hosts. We conclude that Treg naturally exert a negative feedback mechanism on Th1-type responses induced by mature DCs in vivo.

The putative counterparts of human plasmacytoid pre-dendritic cells (pDCs) have been described in vivo in mouse models and very recently in an in vitro culture system. In this study, we report that large numbers of bone marrow-derived murine CD11c+B220+ pDCs can be generated with Flt3 ligand (FL) as the sole exogenous differentiation/growth factor and that pDC generation is regulated in vivo by FL because FL-deficient mice showed a major reduction in splenic pDC numbers. We extensively analyzed bone marrow-derived CD11c+B220+ pDCs and described their immature APC phenotype based on MHC class II, activation markers, and chemokine receptor level of expression. CD11c+B220+ pDCs showed a nonoverlapping Toll-like receptor pattern of expression distinct from that of classical CD11c+B220− dendritic cells and were poor T cell stimulators. Stimulation of CD11c+B220+ pDCs with oligodeoxynucleotides containing certain CpG motifs plus CD40 ligand plus GM-CSF led to increased MHC class II, CD80, CD86, and CD8α expression levels, to a switch in chemokine receptor expression that affected their migration, to IFN-α and IL-12 secretion, and to the acquisition of priming capacities for both CD4+ and CD8+ OVA-specific TCR-transgenic naive T cells. Thus, the in vitro generation of murine pDCs may serve as a useful tool to further investigate pDC biology as well as the potential role of these cells in viral immunity and other settings.

During murine schistosomiasis, there is a gradual switch from a predominant Th1 cytokine response to a Th2-dominated response after egg laying, an event that favors the formation of granuloma around viable eggs. Egg-derived glycoconjugates, including glycolipids, may play a crucial role in this phenomenon. In this study, we used a model of dendritic cell sensitization to study the role of egg glycoconjugates in the induction of specific immune response to soluble egg Ag (SEA) and to investigate the possibility that CD1d, a molecule implicated in glycolipid presentation, may be involved in such a phenomenon. We show that, when captured, processed, and presented to naive T lymphocytes by dendritic cells, egg, but not larval, Ag skew the immune response toward a Th2 response. Periodate treatment reversed this effect, indicating that the sugar moiety of SEA is important in this phenomenon. Using DC treated ex vivo with a neutralizing anti-CD1d Ab or isolated from CD1d knockout mice, we show that CD1d is crucial in the priming of SEA-specific Th2 lymphocytes. We then evaluated the contribution of CD1d on the development of the SEA-specific immune response and on the formation of the egg-induced liver granuloma during murine schistosomiasis. We find that CD1d knockout mice have a reduced Th2 response after egg laying and develop a less marked fibrotic pathology compared with wild-type mice. Altogether, our results suggest that Ag presentation of parasite glycoconjugates to CD1d-restricted T cells may be important in the early events leading to the induction of Th2 responses and to egg-induced pathology during murine schistosomiasis.

Type I IFN are immune modulatory cytokines that are secreted during early stages of infection. Type I IFN bridge the innate and the adaptive immune system in humans and mice. We compared the capacity of type I and II IFN to induce the functional maturation of monocyte-derived dendritic cells (MoDC). Extending our earlier observation that type I IFN promote DC maturation, we report that these cytokines also enhance DC differentiation by augmenting CD40 ligand (CD40L)-induced cytokine secretion by MoDC. Type I IFN alone were poor inducers of MoDC maturation as compared with other stimuli. They up-regulated the expression of HLA-DR, CD80, CD86, partially CCR7 but not CD83, partially reduced antigen-uptake function, increased the levels of IL-12p35 mRNA, and prolonged surface expression of peptide-MHC class I complexes for presentation to cytotoxic T lymphocytes, but did not induce migration towards CCL21 chemokine. However, type I IFN were potent co-factors for CD40L-mediated function. Here, they enhanced CD40L-mediated IL-6, IL-10 and IL-12p70 secretion. Furthermore, when combined with IL-1beta and/or IL-4, IFN-alpha2a type I IFN increased CD40L-mediated IL-12p70 production by 2- to 3-fold, and biased the IL-12 p40/p70 ratio towards the IFN-gamma inducing p70 heterodimer, this correlating with higher levels of IFN-gamma secretion by allogeneic T cell subsets and NK cells. Our results suggest that the rapid expression of CD40L, IFN and IL-1beta at sites of infection and inflammation can act in concert on immature DC, thereby linking innate and adaptive immune responses. In this way, type I IFN play a dual role as DC maturation factors and enhancers of CD40L-mediated DC activation.

Flt3 ligand (FL) and granulocyte-macrophage colony-stimulating factor (GM-CSF) are important growth factors for dendritic cells (DC). Substantial numbers of DC can be generated in vivo following the administration of either factor. We sought to extend our knowledge of the functional properties of these cells including their ability to prime naïve CD8(+) T cells. In addition, we compared the nature of the DC generated in vivo with the single cytokines to those generated with the combination of FL+polyethylene glycol-modified GM-CSF (pGM-CSF). Treatment with FL+pGM-CSF yielded greater numbers of both CD11b(low) and CD11b(high) DC than with either cytokine alone, and these DC were more efficient at antigen (Ag) capture. The FL+pGM-CSF-generated CD11b(low) DC lacked expression of CD8alpha. Following treatment with LPS in vivo, all DC subsets upregulated CD40, CD80, CD86, and MHC class II expression, but surprisingly Ag capture was not downregulated and some DC subsets retained expression of intracellular MHC class II vesicles. Thus, even after activation in vivo with LPS, DC retained Ag capture properties of immature DC, and Ag presentation/costimulation properties of mature DC. Though all DC subsets stimulated CD4(+) T cell proliferation equivalently, FL-generated DC were more efficient at priming Ag-specific CD8(+) cytolytic T cells than DC generated with either pGM-CSF alone or FL+pGM-CSF, and CD11b(high) DC were more efficient at priming CD8(+) T cells than CD11b(low) DC.

Dendritic cells (DCs) are essential for the priming of immune responses. This antigen-presenting function of DCs develops in sequence in a process called maturation, during which they become potent sensitizers of naïve T cells but reduce their ability to capture and process antigens. Some heterogeneity exists in mouse-DC populations, and two distinct subsets of DCs expressing high levels of CD11c can be identified on the basis of CD8α expression. We have studied the phenotype and maturation state of mouse splenic CD8α− and CD8α+ DCs. Both subsets were found to reside in the spleen as immature cells and to undergo a phenotypic maturation upon culture in vitro in GM-CSF-containing medium or in vivo in response to lipopolysaccharide. In vitro and in vivo analyses showed that this maturation process is an absolute requisite for DCs to acquire their T-cell priming capacity, transforming CD8α− and CD8α+ DCs into potent and equally efficient activators of naïve CD4+ and CD8+ T cells. Furthermore, these results highlight the importance that environmental factors may have on the ability of DC subsets to influence Th responses qualitatively; i.e., the ability to drive Th1 versus Th2 differentiation may not be fixed immutably for each DC subset.

Inflammatory mediators facilitate the maturation of dendritic cells (DC), enabling them to induce the activation, proliferation and differentiation of cognate T cells. The role of CD40 on DC and CD154 on T cells has been studied by the co-adoptive transfer of antigen-pulsed DC and TCR-transgenic (Tg) T cells in vivo. It is shown that in the absence of CD40-CD154 interactions, initial Tg T cell expansion occurs in vivo, but over time, T cell expansion cannot be sustained. The basis for the demise of the T cell population is likely due to the disappearance of the antigen-pulsed DC in the draining lymph nodes when CD154-CD40 interactions are interrupted. These findings show that both T cell and DC persistence in vivo is dependent on CD40-CD154 interactions. In addition to the physical persistence of the DC, CD40 triggering of DC also greatly increases the period for which they can productively present antigen to Tg T cells. Hence DC persistence and antigen-presenting cell capacity are both dependent on CD40 signaling. While TNF-alpha can mature DC as measured by a variety of criteria, the unique capacity of CD40 signaling to sustain T cell responses and induce DC maturation is underscored by the inability of TNF-alpha to rescue the immune deficiency of CD40(-/-) DC. Hence, the profound impact of CD154 deficiency on cell-mediated immunity may be due to its ability to limit the duration of antigen presentation in vivo and cause the premature demise of antigen-specific T cells.

Dendritic cells are pivotal antigen-presenting cells for generating adaptive T-cell responses. Here, we show that dendritic cells belonging to either the myeloid-related or lymphoid-related subset are permissive for infection by mouse polyomavirus and, when loaded with a peptide corresponding to the immunodominant anti-polyomavirus CD8+T-cell epitope or infected by polyomavirus, are each capable of driving expansion of primary polyomavirus-specific CD8+T-cell responses in vivo.

Murine dendritic cells (DCs) can be classified into at least 2 subsets, “myeloid-related” (CD11bbright, CD8α−) and “lymphoid-related” (CD11bdull, CD8α+), but the absolute relationship between the 2 remains unclear. Methods of generating DCs from bone marrow (BM) precursors in vitro typically employ granulocyte-macrophage colony-stimulating factor (GM-CSF) as the principal growth factor, and the resultant DCs exhibit a myeloidlike phenotype. Here we describe a flt3-ligand (FL)–dependent BM culture system that generated DCs with more diverse phenotypic characteristics. Murine BM cells cultured at high density in recombinant human FL for 9 days developed into small lymphoid-sized cells, most of which expressed CD11c, CD86, and major histocompatibility complex (MHC) class II. The CD11c+ population could be divided into 2 populations on the basis of the level of expression of CD11b, which may represent the putative myeloid- and lymphoid-related subsets. The FL in vitro–derived DCs, when treated with interferon-α or lipopolysaccharide during the final 24 hours of culture, expressed an activated phenotype that included up-regulation of MHC class II, CD1d, CD8α, CD80, CD86, and CD40. The FL-derived DCs also exhibited potent antigen-processing and antigen-presenting capacity. Neutralizing anti–interleukin-6 (IL-6) antibody, but not anti–GM-CSF, significantly reduced the number of DCs generated in vitro with FL, suggesting that IL-6 has a role in the development of DCs from BM precursors. Stem cell factor, which exhibits some of the same bioactivities as FL, was unable to replace FL to promote DC development in vitro. This culture system will facilitate detailed analysis of murine DC development.

Dendritic cells (DCs) represent a family of ontogenically distinct leukocytes involved in immune response regulation. The ability of DCs to stimulate T-cell immunity has led to their use as vectors for immunotherapy vaccines. However, it is unclear whether and to what degree in vitro–generated DCs are representative of DCs that develop in vivo. Treatment of mice with human Flt3 ligand (FL) dramatically increases the number of DCs. We report here that administration of FL to healthy human volunteers increased the number of circulating CD11c+ IL-3Rlow DC (mean 44-fold) and CD11c− IL-3Rhigh DC precursors (mean 12-fold). Moreover, the CD11c+ DCs were efficient stimulators of T cells in vitro. Thus, FL can expand the number of circulating, functionally competent human DCs in vivo.

Dendritic cells (DC) are potent APCs that can be characterized in the murine spleen as CD11bhighCD11chigh or CD11blowCD11chigh. Daily injection of mice of Flt3 ligand (FL) into mice transiently expands both subsets of DC in vivo, but the effect of administration of GM-CSF on the expansion of DC in vivo is not well defined. To gain further insight into the role of GM-CSF in DC development and function in vivo, we treated mice with polyethylene glycol-modified GM-CSF (pGM-CSF) which has an increased half-life in vivo. Administration of pGM-CSF to mice for 5 days led to a 5- to 10-fold expansion of CD11bhighCD11chigh but not CD11blowCD11chigh DC. DC from pGM-CSF-treated mice captured and processed Ag more efficiently than DC from FL-treated mice. Although both FL- and pGM-CSF-generated CD11bhighCD11chigh DC were CD8α−, a greater proportion of these DC from pGM-CSF-treated mice were 33D1+ than from FL-treated mice. CD11blowCD11chigh DC from FL-treated mice expressed high levels of intracellular MHC class II. DC from both pGM-CSF- and FL-treated mice expressed high levels of surface class II, low levels of the costimulatory molecules CD40, CD80, and CD86 and were equally efficient at stimulating allogeneic and Ag-specific T cell proliferation in vitro. The data demonstrate that treatment with pGM-CSF in vivo preferentially expands CD11bhighCD11chigh DC that share phenotypic and functional characteristics with FL-generated CD11bhighCD11chigh DC but can be distinguished from FL-generated DC on the basis of Ag capture and surface expression of 33D1.

The ligand for the receptor tyrosine kinase fms-like tyrosine kinase 3 (flt3), also referred to as fetal liver kinase-2 (flk-2), has an important role in hematopoiesis. The flt3 ligand (flt3L) is a growth factor for hematopoietic progenitors and induces hematopoietic progenitor and stem cell mobilization in vivo. In addition, when mice are treated with flt3L immature B cells, natural killer (NK) cells and dendritic cells (DC) are expanded in vivo. To further elucidate the role of flt3L in hematopoiesis, mice lacking flt3L (flt3L−/−) were generated by targeted gene disruption. Leukocyte cellularity was reduced in the bone marrow, peripheral blood, lymph nodes (LN), and spleen. Thymic cellularity, blood hematocrit, and platelet numbers were not affected. Significantly reduced numbers of myeloid and B-lymphoid progenitors were noted in the BM of flt3L−/− mice. In addition a marked deficiency of NK cells in the spleen was noted. DC numbers were also reduced in the spleen, LN, and thymus. Both myeloid-related (CD11c++ CD8−) and lymphoid-related (CD11c++ CD8+) DC numbers were affected. We conclude that flt3L has an important role in the expansion of early hematopoietic progenitors and in the generation of mature peripheral leukocytes.

Endothelial cell CD39/ecto-ADPase plays a major role in vascular homeostasis. It rapidly metabolizes ADP released from stimulated platelets, thereby preventing further platelet activation and recruitment. We recently developed a recombinant, soluble form of human CD39, solCD39, with enzymatic and biological properties identical to CD39. To identify amino acids essential for enzymatic/biological activity, we performed site-directed mutagenesis within the four highly conserved apyrase regions of solCD39. Mutation of glutamate 174 to alanine (E174A) and serine 218 to alanine (S218A) resulted in complete and approximately 90% loss of solCD39 enzymatic activity, respectively. Furthermore, compared to wild-type, S57A exhibited a 2-fold increase in ADPase activity without change in ATPase activity, while the tyrosine 127 to alanine (Y127A) mutant lost 50-60% of both ADPase and ATPase activity. The ADPase activity of wild-type solCD39 and each mutant, except for R135A, was greater with calcium as the required divalent cation than with magnesium, but for ATPase activity generally no such preference was observed. Y127A demonstrated the highest calcium/magnesium ADPase activity ratio, 2.8-fold higher than that of wild-type, even though its enzyme activity was greatly reduced. SolCD39 mutants were further characterized by correlating enzymatic with biological activity in an in vitro platelet aggregation system. Each solCD39 mutant was similar to wild-type in reversing platelet aggregation, except for E174A and S218A. E174A, completely devoid of enzymatic activity, failed to inhibit platelet responsiveness, as anticipated. S218A, with 91% loss of ADPase activity, could still reverse platelet aggregation, albeit much less effectively than wild-type solCD39. Thus, glutamate 174 and serine 218 are essential for both the enzymatic and biological activity of solCD39.

CD8+T cells are critical for the clearance of acute polyomavirus infection and the prevention of polyomavirus-induced tumors, but the antigen-presenting cell(s) involved in generating polyomavirus-specific CD8+T cells have not been defined. We investigated whether dendritic cells and macrophages are permissive for polyomavirus infection and examined their potential for inducing antiviral CD8+T cells. Although dendritic cells and macrophages both supported productive polyomavirus infection, dendritic cells were markedly more efficient at presenting the immunodominant viral epitope to CD8+T cells. Additionally, infected dendritic cells, but not infected macrophages, primed anti-polyomavirus CD8+T cells in vivo. Treatment with Flt3 ligand, a hematopoietic growth factor that dramatically expands the number of dendritic cells, markedly enhanced the magnitude of virus-specific CD8+T-cell responses during acute infection and the pool of memory anti-polyomavirus CD8+T cells. These findings suggest that virus-infected dendritic cells induce polyomavirus-specific CD8+T cells in vivo and raise the potential for their use as cellular adjuvants to promote CD8+T cell surveillance against polyomavirus-induced tumors.

An abrogation of the decline in epidermal Langerhans cell numbers above melanoma might significantly improve the efficacy of immunotherapy for melanoma treatment. Systemic Flt3 ligand (FL) administration in mice induced a significant increase in mature dendritic cells (DC) within the skin, preferentially in the dermis, whereas IL-12 promoted a significant increase of immature DC preferentially in the epidermis. Both effects were abrogated in IL-12 knockout mice. Thus, IL-12 could promote FL-induced accumulation of skin DC. The involvement of FL and IL-12 in the regulation of DC accumulation within the skin may contribute, at least in part, to the stimulation of antimelanoma immunity by FL- and IL-12-based immunotherapies. Moreover, FL and IL-12 could be used for selective in vivo generation of DC in either epidermis or dermis for experimental and clinical purposes.

TRAIL (TNF-related apoptosis-inducing ligand) is a member of the TNF family that induces apoptosis in a variety of cancer cells. In this study, we demonstrate that human CD11c(+) blood dendritic cells (DCs) express TRAIL after stimulation with either interferon (IFN)-gamma or -alpha and acquire the ability to kill TRAIL-sensitive tumor cell targets but not TRAIL-resistant tumor cells or normal cell types. The DC-mediated apoptosis was TRAIL specific, as soluble TRAIL receptor blocked target cell death. Moreover, IFN-stimulated interleukin (IL)-3 receptor (R)alpha(+) blood precursor (pre-)DCs displayed minimal cytotoxicity toward the same target cells, demonstrating a clear functional difference between the CD11c(+) DC and IL-3Ralpha(+) pre-DC subsets. These results indicate that TRAIL may serve as an innate effector molecule on CD11c(+) DCs for the elimination of spontaneously arising tumor cells and suggest a means by which TRAIL-expressing DCs may regulate or eliminate T cells responding to antigen presented by the DCs.

Dendritic cells (DCs) are unique in their ability to stimulate T cells and initiate adaptive immunity. Injection of mice with the cytokine Flt3-ligand (FL) dramatically expands mature lymphoid and myeloid-related DC subsets. In contrast, injection of a polyethylene glycol-modified form of granulocyte/macrophage colony-stimulating factor (GM-CSF) into mice only expands the myeloid-related DC subset. These DC subsets differ in the cytokine profiles they induce in T cellsin vivo. The lymphoid-related subset induces high levels of the Th1 cytokines interferon γ and interleukin (IL)-2 but little or no Th2 cytokines. In contrast, the myeloid-related subset induces large amounts of the Th2 cytokines IL-4 and IL-10, in addition to interferon γ and IL-2. FL- or GM-CSF-treated mice injected with soluble ovalbumin display dramatic increases in antigen-specific antibody titers, but the isotype profiles seem critically dependent on the cytokine used. Although FL treatment induces up to a 10,000-fold increase in ovalbumin-specific IgG2a and a more modest increase in IgG1 titers, GM-CSF treatment favors a predominantly IgG1 response with little increase in IgG2a levels. These data suggest that distinct DC subsets have strikingly different influences on the type of immune response generatedin vivoand may thus be targets for pharmacological intervention.

The MHC class I binding proteins leukocyte immunoglobulin-like receptor (LIR)-1 and −2 recognize a similar broad spectrum of HLA-A, -B and -C alleles but are differentially expressed in lymphocytes, monocytes, and dendritic cells. In monocytes, phosphorylation of LIR-1 and LIR-2 results in the binding of the tyrosine phosphatase SHP-1. Coligation of either LIR with Fcγ receptor I (CD64) inhibits tyrosine phosphorylation of the associated Fc receptor γ chain and Syk molecules, as well as intracellular calcium mobilization. These findings suggest that LIR-1 and LIR-2 function as unique MHC class I receptors involved in the inhibition or down-modulation of monocyte activation signals, particularly those mediated through the receptors for IgG, IgE and IgA.

Interleukin-4 contributes to expulsion of certain gastrointestinal parasites and causes intestinal mucosal mastocytosis. Because mast cell-derived mediators are spasmogenic, potentially causing parasitic expulsion, we investigated the effect of interleukin-4 on smooth muscle and the mast cell and mediator dependency of this effect. BALB/c, mast cell-deficient W/Wv mice, 5-lipoxygenase-efficient mice, and their littermate controls were injected with interleukin-4-anti-interleukin-4 antibody complexes that chronically increase serum interleukin-4 levels. Mid-small intestinal segments, hung longitudinally in organ baths, were stimulated electrically or by agonists. The cholinergic response to electrical field stimulation was significantly increased by interleukin-4 treatment in BALB/c but not W/Wv mice. The enhanced cholinergic contraction was not due to increased acetylcholine responsiveness but was dependent on leukotriene D4, since it was reversed by leukotriene D4 receptor antagonism, and not observed in 5-lipoxygenase knock-out mice. Leukotriene D4 responsiveness was unaffected by interleukin-4 treatment. We conclude that interleukin-4 amplifies cholinergic excitation through a mast cell and leukotriene D4-dependent mechanism.

We previously demonstrated that when platelets are in motion and in proximity to endothelial cells, they become unresponsive to agonists (Marcus, A.J., L.B. Safier, K.A. Hajjar, H.L. Ullman, N. Islam, M.J. Broekman, and A.M. Eiroa. 1991. J. Clin. Invest. 88:1690-1696). This inhibition is due to an ecto-ADPase on the surface of endothelial cells which metabolizes ADP released from activated platelets, resulting in blockade of the aggregation response. Human umbilical vein endothelial cells (HUVEC) ADPase was biochemically classified as an E-type ATP-diphosphohydrolase. The endothelial ecto-ADPase is herein identified as CD39, a molecule originally characterized as a lymphoid surface antigen. All HUVEC ecto-ADPase activity was immunoprecipitated by monoclonal antibodies to CD39. Surface localization of HUVEC CD39 was established by confocal microscopy and flow cytometric analyses. Transfection of COS cells with human CD39 resulted in both ecto-ADPase activity as well as surface expression of CD39. PCR analyses of cDNA obtained from HUVEC mRNA and recombinant human CD39 revealed products of the same size, and of identical sequence. Northern blot analyses demonstrated that HUVEC express the same sized transcripts for CD39 as MP-1 cells (from which CD39 was originally cloned). We established the role of CD39 as a prime endothelial thromboregulator by demonstrating that CD39-transfected COS cells acquired the ability to inhibit ADP-induced aggregation in platelet-rich plasma. The identification of HUVEC ADPase/CD39 as a constitutively expressed potent inhibitor of platelet reactivity offers new prospects for antithrombotic therapeusis.

To perform mechanistic studies on the biology of bacteria including metabolism, physiology, and pathogenesis, it is essential to possess the tools required for genetic manipulation. Introduction of plasmid DNA into Staphylococcus epidermidis for subsequent genetic manipulation, including allelic replacement and complementation experiments, is typically performed by either electroporation or conjugative mobilization. Herein, standard protocols and tips for the transfer of plasmid DNA to S. epidermidis by these two methods are provided.

IFN-gamma has been shown to either stimulate or inhibit Ig secretion. No studies have yet addressed the basis for these seemingly conflicting properties nor whether IFN-gamma acted directly at the level of the B cell to mediate its effects. Thus, we studied the ability of IFN-gamma to regulate Ig secretion in sort-purified, resting murine B cells that were99% Ig+, activated either through membrane Ig using unconjugated or dextran-conjugated anti-IgD antibodies (alphadelta-dex) or through CD40 using soluble or membrane CD40 ligand (CD40L). B cells activated with alphadelta-dex proliferated but do not secrete Ig, even in the presence of IL-1 + IL-2. We demonstrate that IFN-gamma only when added subsequent to B cell stimulation with alphadelta-dex, but not unconjugated anti-IfD antibody, plus IL-1 + IL-2 induces up to 100-fold enhancements in Ig secretion and in the numbers of Ig-secreting cells. The predominant Ig isotype secreted is IgM, with IgG3 and IgG2a comprising the majority of non-IgM antibody. IFN-gamma must act in concert with IL-2 for stimulation of Ig secretion. Further, IFN-gamma synergizes with IL-3 + granulocyte-macrophage colony stimulating factor for induction of Ig synthesis. IFN-gamma also enhances IgA syntheses by transforming growth factor-beta-induced membrane IgA+ cells. By contrast, 125IIFN-gamma fails to stimulate Ig secretion in B cells activated with CD40L in the presence or absence of IL-1 + IL-2 or IL-4. However, the combination of CD40L and alphabeta-dex is strongly synergistic for IFN-gamma-induced Ig secretion. Thus, these data establish that IFN-gamma can act directly on the B cell to induce Ig synthesis without the participation of any other cell and demonstrates that the mode of activation of the B cell plays an important role in directing the action of IFN-gamma.

Mutations in the gene encoding the protein tyrosine kinase Btk are associated with the human B cell immunodeficiency X-linked agammaglobulinemia (XLA). In the mouse, a point mutation in the Btk pleckstrin homology domain segregates with a milder X-linked immunodeficiency (xid). To assessthe importance of Btk function in murine lymphopoiesis, we generated multiple embryonic stem cell clones bearing a targeted disruption of the btk gene and examined their potential to produce lymphocytes in both C57BL/6 and RAG2−/−host chimeric animals. These mice provide a complementary set of in vivo competition assays that formally establish the genetic basis for the xid phenotype. Although the null mutation yields a phenotype quite similar to that of xid, it also compromises expansion of B cell precursors. Our results suggest that the murine and human consequences of Btk deficiency differ only quantitatively, and represent the same disease process.

A ligand for CD30 has been recently cloned, and has been shown to have sequence homology with the tumor necrosis factor family of cytokines. CD30 ligand (CD30L) was found to be induced on helper T cell clones, and its receptor was expressed on freshly isolated and activated murine B cells. Recombinant murine CD30L was found to share many functional properties with CD40 ligand (CD40L) in the regulation of murine B cell growth and differentiation in vitro. CD30L stimulated B cell proliferation, antigen-specific antibody production, and polyclonal immunoglobulin secretion in a cytokine-dependent manner. In particular, the stimulation of B cell proliferation by CD30L required interleukin (IL)-4 and IL-5, induction of anti-sheep red blood cell antibody-secreting B cells by CD30L required IL-2 and IL-5, and optimal induction of polyclonal immunoglobulin secretion required IL-4 and IL-5. Under these conditions, the polyclonal secretion of IgG1, IgA, IgG3 and IgE was induced. The induction of immunoglobulin secretion by CD30L was independent of CD40L, as B cells from CD40L deficient-mice responded normally to CD30L treatment. We conclude that CD30L is a potent mediator of B cell growth and differentiation in vitro and may play a role in cognate T cell-B cell interactions.

Individuals with X-linked hyper-IgM syndrome fail to express functional CD40 ligand (CD40L) and, as a consequence, are incapable of mounting protective antibody responses to opportunistic bacterial infections. To address the role of CD40L in humoral immunity, we created, through homologous recombination, mice deficient in CD40L expression. These mice exhibited no gross developmental deficiencies or health abnormalities and contained normal percentages of B and T cell subpopulations. CD40L-deficient mice did display selective deficiencies in humoral immunity; basal serum isotype levels were significantly lower than observed in normal mice, and IgE was undetectable. Furthermore, the CD40L-deficient mice failed to mount secondary antigen-specific responses to immunization with a thymus-dependent antigen, trinitrophenol-conjugated keyhole limpet hemocyanin (TNP-KLH). By contrast, the CD40L-deficient mice produced antigen-specific antibody of all isotypes except IgE in response to the thymus-independent antigen, DNP-Ficoll. These results underscore the requirement of CD40L for T cell-dependent antibody responses. Moreover, Ig class switching to isotypes other than IgE can occur in vivo in the absence of CD40L, supporting the notion that alternative B cell signaling pathways regulate responses to thymus-independent antigens.

Most inbred strains of mouse infected with the intestinal nematode Trichuris muris are resistant to infection expelling the parasite before adult worms establish. However, a few susceptible strains exist that are incapable of worm expulsion and harbor chronic infections of mature adult worms. Analyses of in vitro cytokine production by cells from the draining lymph node (mesenteric lymph node) have indicated that expulsion phenotype is tightly correlated with the selective expansion of helper T cells (Th) of the Th1 or Th2 cell subset within the mesenteric lymph node, resulting in susceptibility and resistance to T. muris, respectively. We have now confirmed and extended our in vitro observations in a series of experiments involving the in vivo manipulation of host cytokine levels. Depletion of interferon (IFN)-gamma in normally susceptible mice resulted in expulsion of the parasite, representing the first evidence for a role for IFN-gamma in the establishment of chronic helminth infection. Blocking interleukin (IL)-4 function in normally resistant animals prevented the generation of a protective immune response allowing adult stages of the parasite to develop. Conversely the administration of IL-4 to a normally susceptible host facilitated expulsion and indeed enabled established adult worms to be expelled when administered late in infection. In all cases assessment of a variety of in vivo parameters indicative of a Th1- or Th2-type response (parasite-specific immunoglobulin (Ig) G2a and the parasite-specific IgG1, total IgE levels and intestinal mastocytosis, respectively) demonstrated that the in vivo modulation of a Th1- or Th2-specific cytokine allowed the reciprocal Th cell subset to expand and become dominant with dramatic consequences for worm expulsion.

We previously described the cloning of a human myeloid cell surface receptor for the Fc region of immunoglobulin A (Fc alpha R). In the present study, a soluble version of the Fc alpha R (solFc alpha R) was generated by removing the transmembrane and cytoplasmic coding regions from full-length Fc alpha R cDNA and ligating into a mammalian expression vector. COS-7 cells transfected with the solFc alpha R plasmid secreted a protein that inhibited both immunoglobulin A (IgA) and anti-Fc alpha R monoclonal antibody (mAb) binding to Fc alpha R+ U937 cells. Furthermore, the solFc alpha R bound specifically to and could be eluted from an anti-Fc alpha R mAb-immunoaffinity column, retaining biological activity. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that the recombinant full-length Fc alpha R migrates over a molecular mass range of approximately 40-60 kd, consistent with the reported size and heterogeneity of the naturally occurring myeloid cell surface Fc alpha R. The solFc alpha R ran on SDS-PAGE as a smaller band (37-55 kd) that reduced to two bands of 23 and 25 kd following N-glycanase treatment, indicating that the Fc alpha R is a heavily glycosylated protein. The biochemical data, coupled with flow cytometry studies showing that the recombinant Fc alpha Rs bind to five different anti-Fc alpha R mAbs, clearly demonstrate that the cloned Fc alpha R corresponds directly to the major Fc alpha R species expressed on human monocytes, neutrophils, and myeloid cell lines. The generation of soluble receptor protein will permit investigations of the role of Fc alpha R in IgA-mediated immunoregulation, effector functions, and disease.

We have investigated the in vitro effects of the cytokine leukemia inhibitory factor (LIF) on normal murine hematopoietic progenitors by measuring recovery and retroviral vector infection efficiency of 13-day posttransplant, spleen-colony-forming cell (CFU-S 13) in short-term culture. Up to a twofold increase in CFU-S13 recovery was observed, from 9.7 x 10(-5) cells in untreated controls to 17.8 to 19.5 x 10(-5) cells, depending on the concentration of LIF. Histologic analysis of spleen colonies from control and LIF-treated marrows demonstrated that there was no detectable alteration in the differentiative potential of CFU-S13. The efficiency of CFU-S13 infection was increased from 15% in untreated controls to 84% to 91% in LIF-treated marrows. Analysis of proviral integration sites in spleen colonies indicated that some CFU- S13 precursors were infected in the LIF-treated marrows.

IgA, the predominant isotype in secretions, mediates the neutralization and removal of environmental antigens from mucosal sites. Although cell surface receptors for the Fc region of IgA (Fc alpha R) have been implicated in a variety of immune effector mechanisms, the molecular features of Fc alpha R remain only marginally characterized. In this report, we describe the isolation of a clone from a myeloid cell line cDNA library that directs the expression of a cell surface molecule with IgA binding specificity. The cDNA encodes a peptide of Mr 30,000 including a putative transmembrane region with features atypical of conventional membrane-anchored proteins. Databank searches indicate that the human myeloid cell Fc alpha R sequence is unique, is a member of the immunoglobulin gene superfamily, and is related to Fc receptors for IgG (Fc gamma RI, II, and III) and IgE (Fc epsilon RI).

Introduction: This study evaluated CD39 in a porcine model of balloon angioplasty and in plasma of patients undergoing percutaneous intervention. CD39 (E-NTPDase1), is the endothelial ecto-ADPase inhibiting platelet function via hydrolysis of released platelet ADP. Methods and Results: A recombinant soluble form of CD39 (solCD39) given intravenously to pigs had an elimination half life of 5–7 days, increased the bleeding time to an extent similar to aspirin, and inhibits platelet aggregation by > 90%. Platelet counts and clot retraction remained normal following solCD39 administration. In a pig model of acute coronary balloon injury, solCD39 resulted in non-statistically significant decreases in platelet (7.7 ± 1.4 versus 11.7 ± 3.4) and fibrin (3.5 ± 0.4 versus 4.2 ± 0.7) deposition ratios. Adding ex vivo to human platelet rich plasma (PRP) solCD39 produced nearly 100% inhibition of ADP-induced platelet aggregation. A dose-response effect of solCD39 on platelet aggregation induced by collagen or a thrombin receptor activating peptide (TRAPSFLLRN) was noted in PRP obtained from volunteers and patients receiving aspirin, clopidogrel or ticlopidine. SolCD39 also provided additional and complete inhibition of TRAP-induced platelet aggregation in PRP from patients who had received abciximab, aspirin and clopidogrel. Conclusions: SolCD39, a novel inhibitor of platelet activation and recruitment with a relatively long half-life appears to be well tolerated and is a potent inhibitor of ADP-, collagen-, or TRAP-induced platelet activation. Its potential use in percutaneous coronary intervention requires further study. Abbreviated Abstract. E-NTPDase1/CD39 is the endothelial ecto-ADPase responsible for inhibition of platelet function. A recombinant soluble form (solCD39) had an elimination half life of 5–7 days in pigs, elevated bleeding times similar to aspirin, did not affect clot retraction, and inhibited platelet aggregation by > 90%. When combined with standard heparin therapy in a pig model of acute coronary balloon injury, solCD39 resulted in a trend toward a decrease in platelet and fibrin deposition. SolCD39 added ex vivo to human platelet rich plasma yielded nearly 100% inhibition of ADP-induced platelet aggregation and provided further inhibition when combined with standard therapy.