Effect of a Lactococcus lactis culture supernatant on diarrhea and performance parameters of piglets in the post-weaning period and on expression of the faeG gene in vitro.

Objectives: To evaluate the effects of a cell-free supernatant from Lactococcus lactis (CFSM) on performance and diarrhearelated parameters and the presence of F4+ enterotoxigenic E. coli (ETEC) in piglets during post-weaning, and to evaluate the in vitro effect of the CFSM on faeG gene expression in an E. coli F4+. Animals and procedure: In 3 trials with 90 piglets per trial, pigs were assigned to receive a placebo or 1 of 2 CFSM treatments and observed for diarrhea and performance. Fecal swabs were taken to determine the presence of ETEC. Quantitative RT-PCR was used to assess faeG gene expression in E. coli 21259 after treatment with CFSM at 50 mg/mL. Results: The CFSM administered for 14 d at a dose of 24 mg/kg BW (2X) reduced diarrhea-related parameters compared to the placebo. Quantitative RT-PCR showed that, in E. coli 21259 treated with CFSM at 50 mg/mL, expression of the faeG gene was significantly repressed (P < 0.0001) relative to that in the untreated control. Conclusion: The evaluated CFSM reduced the frequency and prevalence of diarrhea in a field situation. The in vitro treatment had an inhibitory effect on the expression of the faeG gene in F4+ E. coli 21259.

Effet d'un surnageant de culture de Lactococcus lactis sur la diarrhée et les paramètres de performance des porcelets en période post-sevrage et sur l'expression du gène faeG in vitro. Objectifs: Évaluer les effets d'un surnageant acellulaire de Lactococcus lactis (CFSM) sur les paramètres de performance et de diarrhée et la présence d'E. coli entérotoxinogène F4+ (ETEC) chez les porcelets en post-sevrage, et évaluer l'effet in vitro du CFSM sur l'expression du gène faeG dans un E. coli F4+. Animaux et procédure: Dans 3 essais portant sur 90 porcelets par essai, les porcs ont reçu un placebo ou 1 des 2 traitements CFSM et ont été observés pour détecter la diarrhée et leurs performances. Des prélèvements fécaux ont été effectués pour déterminer la présence d'ETEC. La RT-PCR quantitative a été utilisée pour évaluer l'expression du gène faeG dans E. coli 21259 après traitement avec CFSM à 50 mg/mL. Résultats: Le CFSM administré pendant 14 jours à une dose de 24 mg/kg de poids corporel (2X) a réduit les paramètres liés à la diarrhée par rapport au placebo. La RT-PCR quantitative a montré que, chez E. coli 21259 traité avec CFSM à 50 mg/mL, l'expression du gène faeG était significativement réprimée (P < 0,0001) par rapport à celle du témoin non traité. Conclusion: Le CFSM évalué a réduit la fréquence et la prévalence de la diarrhée sur le terrain. Le traitement in vitro a eu un effet inhibiteur sur l'expression du gène faeG chez F4+ E. coli 21259.(Traduit par Dr Serge Messier).

Evaluation of a Lactococcus lactis-based dried fermentation product administered through drinking water on nursery pig growth performance, fecal Escherichia coli virulence genes and pathotypes, antibiotic usage, and mortality.

A total of 34,749 pigs were used in two experiments to evaluate the effects of a postbiotic dried fermentation product (DFP) administered through drinking water on nursery pig growth performance, antibiotic injection frequency, morbidity, mortality, fecal consistency, and characterization of fecal Escherichia coli. The DFP is composed of bioactive molecules derived from Lactococcus lactis. In Exp. 1, 350 barrows (DNA Line 200 × 400; initial body weight [BW] 6.1 ± 0.01 kg) were used in a 42-d study with five pigs per pen and 35 pens per treatment. The DFP was supplied for 14 d at a target dosage of 24 mg/kg BW using a water medicator at a 1:128 dilution. On days 7 and 14, fecal samples were collected for dry matter (DM) and to determine, by a multiplex polymerase chain reaction (PCR) assay, prevalence of 11 virulence genes characteristic of E. coli pathotypes. There was no evidence (P > 0.10) for differences for growth, incidence of diarrhea, number of antibiotic injections, removals, or fecal DM. On both fecal collection days, E. coli virulence genes were present with day 7 samples positive for genes that encode for hemolysins (hlyA, exhA), intimin (eae), and enteroaggregative heat-stable enterotoxin (astA). Prevalence of enterotoxin genes (elt, estA, estB, astA) increased on day 14, but DFP had no effects on the prevalence of any of the virulence genes. A total of 32 out of 72 E. coli isolates were identified as enterotoxigenic pathotype and all except one were from day 14 fecal samples. Fourteen isolates were positive for F4 fimbria and one isolate was positive for F4 and F18 fimbriae. In Exp. 2, 34,399 nursery pigs (initially 5.6 kg) were used in 20 nursery barns with 10 barns per treatment (control or DFP). The target dosage of the DFP for the first 14 d was 35 mg/kg BW. Following the 14-d supplementation period, pigs continued to be monitored for approximately 31 d. There was no evidence (P > 0.05) for the DFP to influence the overall percentage of pigs that died or growth performance. From days 0 to 14, providing the DFP reduced (P < 0.05) the percentage of pigs that were euthanized. However, providing the DFP increased (P < 0.05) the overall percentage of pigs that were euthanized and total mortality. For the number of antibiotic injections (treatment interventions), providing the DFP reduced the number of injections for the common period (P < 0.001) and overall (P = 0.002). These results indicate that the DFP did not influence growth performance but providing the DFP in Exp. 2 led to increased total nursery pig mortality.

Probiotic disruption of quorum sensing reduces virulence and increases cefoxitin sensitivity in methicillin-resistant Staphylococcus aureus.

Therapies which target quorum sensing (QS) systems that regulate virulence in methicillin-resistant Staphylococcus aureus (MRSA) are a promising alternative to antibiotics. QS systems play a crucial in the regulation of MRSA antibiotic resistance, exotoxin production, antioxidant protection and immune cell evasion, and are therefore attractive therapeutic targets to reduce the virulence of a pathogen. In the present work the the effects of bioactive peptides isolated from two strains of lactic acid bacteria were tested against antibiotic resistance, carotenoid production, resistance to oxidative killing and biofilm structure in two clinical MRSA isolates. The results obtained from fractional-inhibitory concentration assays with bulk and semi-purified bioactive molecules showed a significant synergistic effect increasing cefoxitin mediated killing of MRSA. This was coupled to a six-fold decrease of the major membrane pigment staphyloxanthin, and a 99% increase in susceptibility to oxidative stress mediated killing. Real-time quantitative PCR analysis of the QS-genes agrA and luxS, showed differential expression between MRSA strains, and a significant downregulation of the hemolysin gene hla. Light microscopy and scanning electron microscopy revealed alteration in biofilm formation and clustering behavior. These results demonstrate that bioactive metabolites may be effectively applied in tandem with beta-lactam antibiotics to sensitize MRSA to cefoxitin. Moreover, these results shown that several key QS-controlled virulence mechanisms are diminished by probiotic metabolites.

Changes in bile acids, FGF-19 and sterol absorption in response to bile salt hydrolase active L. reuteri NCIMB 30242.

The size and composition of the circulating bile acid (BA) pool are important factors in regulating the human gut microbiota. Disrupted regulation of BA metabolism is implicated in several chronic diseases. Bile salt hydrolase (BSH)-active Lactobacillus reuteri NCIMB 30242, previously shown to decrease LDL-cholesterol and increase circulating BA, was investigated for its dose response effect on BA profile in a pilot clinical study. Ten otherwise healthy hypercholesterolemic adults, recruited from a clinical trial site in London, ON, were randomized to consume delayed release or standard release capsules containing L. reuteri NCIMB 30242 in escalating dose over 4 weeks. In another aspect, 4 healthy normocholesterolemic subjects with LDL-C below 3.4 mmol/l received delayed release L. reuteri NCIMB 30242 at a constant dose over 4 weeks. The primary outcome measure was the change in plasma BA profile over the intervention period. Additional outcomes included circulating fibroblast growth factor (FGF)-19, plant sterols and LDL-cholesterol as well as fecal microbiota and bsh gene presence. After one week of intervention subjects receiving delayed release L. reuteri NCIMB 30242 increased total BA by 1.13 ± 0.67 µmol/l (P = 0.02), conjugated BA by 0.67 ± 0.39 µmol/l (P = 0.02) and unconjugated BA by 0.46 ± 0.43 µmol/l (P = 0.07), which represented a greater than 2-fold change relative to baseline. Increases in BA were largely maintained post-week 1 and were generally correlated with FGF-19 and inversely correlated with plant sterols. This is the first clinical support showing that a BSH-active probiotic can significantly and rapidly influence BA metabolism and may prove useful in chronic diseases beyond hypercholesterolemia.

Bacterial bile metabolising gene abundance in Crohn's, ulcerative colitis and type 2 diabetes metagenomes.

We performed an analysis to determine the importance of bile acid modification genes in the gut microbiome of inflammatory bowel disease and type 2 diabetic patients. We used publicly available metagenomic datasets from the Human Microbiome Project and the MetaHIT consortium, and determined the abundance of bile salt hydrolase gene (bsh), 7 alpha-dehydroxylase gene (adh) and 7-alpha hydroxysteroid dehydrogenase gene (hsdh) in fecal bacteria in diseased populations of Crohn's disease (CD), Ulcerative Colitis (UC) and Type 2 diabetes mellitus (T2DM). Phylum level abundance analysis showed a significant reduction in Firmicute-derived bsh in UC and T2DM patients but not in CD patients, relative to healthy controls. Reduction of adh and hsdh genes was also seen in UC and T2DM patients, while an increase was observed in the CD population as compared to healthy controls. A further analysis of the bsh genes showed significant differences in the correlations of certain Firmicutes families with disease or healthy populations. From this observation we proceeded to analyse BSH protein sequences and identified BSH proteins clusters representing the most abundant strains in our analysis of Firmicute bsh genes. The abundance of the bsh genes corresponding to one of these protein clusters was significantly reduced in all disease states relative to healthy controls. This cluster includes bsh genes derived from Lachospiraceae, Clostridiaceae, Erysipelotrichaceae and Ruminococcaceae families. This metagenomic analysis provides evidence of the importance of bile acid modifying enzymes in health and disease. It further highlights the importance of identifying gene and protein clusters, as the same gene may be associated with health or disease, depending on the strains expressing the enzyme, and differences in the enzymes themselves.

Ingestible gastrointestinal sampling devices: state-of-the-art and future directions.

Despite the significant contribution of gastrointestinal diseases to the global disease burden and the increasing recognition of the role played by the intestinal microbiota in human health and disease states, conventional methods of exploring and collecting samples from the gastrointestinal tract remain invasive, resource intensive, and often unable to capture all the information contained in these heterogeneous samples. A new class of gastrointestinal sampling capsules is emerging in the literature, which contains the components required for an autonomous intra-luminal device and preserves the spatial and temporal information of the gastrointestinal samples. In this paper, we identify the primary design requirements for gastrointestinal sampling capsules, and we review the state-of-the-art for different components and functionalities. We also suggest two design concepts, and we highlight future directions for this class of biomedical devices.

Emerging science of the human microbiome.

The human gastrointestinal tract hosts a large number of microbial cells which exceed their mammalian counterparts by approximately 3-fold. The genes expressed by these microorganisms constitute the gut microbiome and may participate in diverse functions that are essential to the host, including digestion, regulation of energy metabolism, and modulation of inflammation and immunity. The gut microbiome can be modulated by dietary changes, antibiotic use, or disease. Different ailments have distinct associated microbiomes in which certain species or genes are present in different relative quantities. Thus, identifying specific disease-associated signatures in the microbiome as well as the factors that alter microbial populations and gene expression will lead to the development of new products such as prebiotics, probiotics, antimicrobials, live biotherapeutic products, or more traditional drugs to treat these disorders. Gained knowledge on the microbiome may result in molecular lab tests that may serve as personalized tools to guide the use of the aforementioned products and monitor interventional progress.

The human microbiome and bile acid metabolism: dysbiosis, dysmetabolism, disease and intervention.

INTRODUCTION: Recent evidence indicates that the human gut microbiome plays a significant role in health and disease. Dysbiosis, defined as a pathological imbalance in a microbial community, is becoming increasingly appreciated as a 'central environmental factor' that is both associated with complex phenotypes and affected by host genetics, diet and antibiotic use. More recently, a link has been established between the dysmetabolism of bile acids (BAs) in the gut to dysbiosis. AREAS COVERED: BAs, which are transformed by the gut microbiota, have been shown to regulate intestinal homeostasis and are recognized as signaling molecules in a wide range of metabolic processes. This review will examine the connection between BA metabolism as it relates to the gut microbiome and its implication in health and disease. EXPERT OPINION: A disrupted gut microbiome, including a reduction of bile salt hydrolase (BSH)-active bacteria, can significantly impair the metabolism of BAs and may result in an inability to maintain glucose homeostasis as well as normal cholesterol breakdown and excretion. To better understand the link between dysbiosis, BA dysmetabolism and chronic degenerative disease, large-scale metagenomic sequencing studies, metatranscriptomics, metaproteomics and metabolomics should continue to catalog functional diversity in the gastrointestinal tract of both healthy and diseased populations. Further, BSH-active probiotics should continue to be explored as treatment options to help restore metabolic levels.

Effect of orally administered L. fermentum NCIMB 5221 on markers of metabolic syndrome: an in vivo analysis using ZDF rats.

Metabolic syndrome, encompassing type 2 diabetes mellitus and cardiovascular disease, is a growing health concern of industrialized countries. Ferulic acid (FA) is a phenolic acid found in foods normally consumed by humans that has demonstrated antioxidant activity, cholesterol-lowering capabilities, and anti-tumorigenic properties. Select probiotic bacteria, including Lactobacillus fermentum NCIMB 5221, produce FA due to intrinsic ferulic acid esterase activity. The aim of the present research was to investigate a FA-producing probiotic, L. fermentum NCIMB 5221, as a biotherapeutic for metabolic syndrome. The probiotic formulation was administered daily for 8 weeks to Zucker diabetic fatty (ZDF) rats, a model of hyperlipidemia and hyperglycemia. Results show that the probiotic formulation reduced fasting insulin levels and insulin resistance, significantly reduced serum triglycerides (p = 0.016), lowered serum low-density lipoprotein cholesterol levels (p = 0.008), and significantly reduced the atherogenic (p = 0.016) and atherosclerosis (p = 0.012) index as compared to the control animals. In addition, the probiotic formulation significantly increased high-density lipoprotein cholesterol levels (p = 0.041) as compared to the control animals. This research indicates that administration of the FA-producing L. fermentum NCIMB 5221 has the potential to reduce insulin resistance, hyperinsulinemia, hypercholesterolemia, and other markers involved in the pathogenesis of metabolic syndrome. Further studies are required to investigate the human clinical potential of the probiotic formulation in affecting the markers and pathogenesis of metabolic syndrome.

In vitro and in vivo characterization and strain safety of Lactobacillus reuteri NCIMB 30253 for probiotic applications.

Lactobacillus reuteri NCIMB 30253 was shown to have potential as a probiotic by reducing the proinflammatory chemokine interleukin-8. Moreover, this strain was evaluated, by in vitro and in vivo techniques, for its safety for human consumption. The identity of the strain was investigated by metabolic profiling and 16S rRNA gene sequencing, and in vitro safety evaluations were performed by molecular and metabolic techniques. Genetic analysis was confirmed by assessing the minimum inhibitory concentration to a panel of antibiotics, showing that the strain was susceptible to 8 antibiotics tested. The ability of the strain to produce potentially harmful by-products and antimicrobial compounds was evaluated, showing that the strain does not produce biogenic amines and does not show bacteriocin activity or reuterin production. A 28-day repeated oral dose study was conducted in normal Sprague-Dawley rats to support the in vivo strain safety. Oral administration of the strain resulted in no changes in general condition and no clinically significant changes to biochemical and haematological markers of safety relative to vehicle control treated animals. This comprehensive assessment of safety of L. reuteri NCIMB 30253 supports the safety of the strain for use as a probiotic.

Novel nitric oxide producing probiotic wound healing patch: preparation and in vivo analysis in a New Zealand white rabbit model of ischaemic and infected wounds.

The treatment of chronic wounds poses a significant challenge for clinicians and patients alike. Here we report design and preclinical efficacy of a novel nitric oxide gas (gNO)-producing probiotic patch for wound healing. Specifically, a wound healing patch using lactic acid bacteria in an adhesive gas permeable membrane has been designed and investigated for treating ischaemic and infected full-thickness dermal wounds in a New Zealand white rabbit model for ischaemic wound healing. Kaplan-Meier survival curves showed increased wound closure with gNO-producing patch-treated wounds over 21 days of therapy (log-rank P = 0·0225 and Wilcoxon P = 0·0113). Cox proportional hazard regression showed that gNO-producing patch-treated wounds were 2·52 times more likely to close compared with control patches (hazard P = 0·0375, score P = 0·032 and likelihood ratio P = 0·0355), and histological analysis showed improved wound healing in gNO-producing patch-treated animals. This study may provide an effective, safe and less costly alternative for treating chronic wounds.

Probiotic Ferulic Acid Esterase Active Lactobacillus fermentum NCIMB 5221 APA Microcapsules for Oral Delivery: Preparation and in Vitro Characterization.

Probiotics possess potential therapeutic and preventative effects for various diseases and metabolic disorders. One important limitation for the oral delivery of probiotics is the harsh conditions of the upper gastrointestinal tract (GIT) which challenge bacterial viability and activity. One proposed method to surpass this obstacle is the use of microencapsulation to improve the delivery of bacterial cells to the lower GIT. The aim of this study is to use alginate-poly-L-lysine-alginate (APA) microcapsules to encapsulate Lactobacillus fermentum NCIMB 5221 and characterize its enzymatic activity and viability through a simulated GIT. This specific strain, in previous research, was characterized for its inherent ferulic acid esterase (FAE) activity which could prove beneficial in the development of a therapeutic for the treatment and prevention of cancers and metabolic disorders. Our findings demonstrate that the APA microcapsule does not slow the mass transfer of substrate into and that of the FA product out of the microcapsule, while also not impairing bacterial cell viability. The use of simulated gastrointestinal conditions led to a significant 2.5 log difference in viability between the free (1.10 × 104 ± 1.00 × 103 cfu/mL) and the microencapsulated (5.50 × 106 ± 1.00 × 105 cfu/mL) L. fermentum NCIMB 5221 following exposure. The work presented here suggests that APA microencapsulation can be used as an effective oral delivery method for L. fermentum NCIMB 5221, a FAE-active probiotic strain.

Ihog and Boi are essential for Hedgehog signaling in Drosophila.

BACKGROUND: The Hedgehog (Hh) signaling pathway is important for the development of a variety of tissues in both vertebrates and invertebrates. For example, in developing nervous systems Hh signaling is required for the normal differentiation of neural progenitors into mature neurons. The molecular signaling mechanism underlying the function of Hh is not fully understood. In Drosophila, Ihog (Interference hedgehog) and Boi (Brother of Ihog) are related transmembrane proteins of the immunoglobulin superfamily (IgSF) with orthologs in vertebrates. Members of this IgSF subfamily have been shown to bind Hh and promote pathway activation but their exact role in the Hh signaling pathway has remained elusive. To better understand this role in vivo, we generated loss-of-function mutations of the ihog and boi genes, and investigated their effects in developing eye and wing imaginal discs. RESULTS: While mutation of either ihog or boi alone had no discernible effect on imaginal tissues, cells in the developing eye disc that were mutant for both ihog and boi failed to activate the Hh pathway, causing severe disruption of photoreceptor differentiation in the retina. In the anterior compartment of the developing wing disc, where different concentrations of the Hh morphogen elicit distinct cellular responses, cells mutant for both ihog and boi failed to activate responses at either high or low thresholds of Hh signaling. They also lost their affinity for neighboring cells and aberrantly sorted out from the anterior compartment of the wing disc into posterior territory. We found that ihog and boi are required for the accumulation of the essential Hh signaling mediator Smoothened (Smo) in Hh-responsive cells, providing evidence that Ihog and Boi act upstream of Smo in the Hh signaling pathway. CONCLUSIONS: The consequences of boi;ihog mutations for eye development, neural differentiation and wing patterning phenocopy those of smo mutations and uncover an essential role for Ihog and Boi in the Hh signaling pathway.

Drosophila glial glutamate transporter Eaat1 is regulated by fringe-mediated notch signaling and is essential for larval locomotion.

In the mammalian CNS, glial cells expressing excitatory amino acid transporters (EAATs) tightly regulate extracellular glutamate levels to control neurotransmission and protect neurons from excitotoxic damage. Dysregulated EAAT expression is associated with several CNS pathologies in humans, yet mechanisms of EAAT regulation and the importance of glutamate transport for CNS development and function in vivo remain incompletely understood. Drosophila is an advanced genetic model with only a single high-affinity glutamate transporter termed Eaat1. We found that Eaat1 expression in CNS glia is regulated by the glycosyltransferase Fringe, which promotes neuron-to-glia signaling through the Delta-Notch ligand-receptor pair during embryogenesis. We made Eaat1 loss-of-function mutations and found that homozygous larvae could not perform the rhythmic peristaltic contractions required for crawling. We found no evidence for excitotoxic cell death or overt defects in the development of neurons and glia, and the crawling defect could be induced by postembryonic inactivation of Eaat1. Eaat1 fully rescued locomotor activity when expressed in only a limited subpopulation of glial cells situated near potential glutamatergic synapses within the CNS neuropil. Eaat1 mutants had deficits in the frequency, amplitude, and kinetics of synaptic currents in motor neurons whose rhythmic patterns of activity may be regulated by glutamatergic neurotransmission among premotor interneurons; similar results were seen with pharmacological manipulations of glutamate transport. Our findings indicate that Eaat1 expression is promoted by Fringe-mediated neuron-glial communication during development and suggest that Eaat1 plays an essential role in regulating CNS neural circuits that control locomotion in Drosophila.

Antimicrobial properties of nitric oxide and its application in antimicrobial formulations and medical devices.

This review describes the antimicrobial properties of nitric oxide (NO) and its application as an antimicrobial agent in different formulations and medical devices. We depict the eukaryotic biosynthesis of NO and its physiologic functions as a cell messenger and as an antimicrobial agent of the cell-mediated immune response. We analyze the antimicrobial activity of NO and the eukaryotic protective mechanisms against NO for the purpose of delineating the therapeutic NO dosage range required for an efficacious and safe antimicrobial activity. We also examine the role of NO produced by virulent bacteria in lessening the efficacy of traditional antimicrobials. In addition, we discuss the efficacy of NO in the healing of infected wounds, describing different NO-producing devices by category, analyzing therapeutic levels, duration of NO production, as well as commercial considerations. Finally, we provide current and future prospects for the design and use of NO-producing devices.

A novel nitric oxide producing probiotic patch and its antimicrobial efficacy: preparation and in vitro analysis.

Microbial and fungal infections are a significant consideration in the etiology of all wounds. Numerous antimicrobial and antifungal formulations have been developed with varying degrees of efficacy and stability. Here, we report a nitric oxide producing probiotic adhesive patch device and investigate its antimicrobial and antifungal efficacy in vitro. This probiotic patch utilizes the metabolic activity of immobilized lactic acid bacteria, glucose, and nitrite salts for the production of gaseous nitric oxide (gNO), which is used as an antimicrobial agent against bacterial and fungal pathogens. Results show that application of gNO-producing probiotic patches to cultures of E. coli, S. aureus, P. aeruginosa, MRSA, T. mentagrophytes, and T. rubrum resulted in complete cell death at between 4 and 8 h, and application to cultures of A. baumannii, resulted in fewer than ten colonies detected per milliliter at 6 h. These results demonstrate that a gNO-producing probiotic patch device containing bacteria, glucose, and nitrite salts can produce sufficient levels of gNO over a therapeutically relevant duration to kill common bacterial and fungal wound pathogens in humans.

IL-12 immunotherapy of murine leukaemia: comparison of systemic versus gene modified cell therapy.

The ability of IL-12 to initiate anti-leukaemia immune responses has been well established; however clinical outcomes fail to recapitulate the therapeutic benefits observed in the laboratory. To address this, we compared two systems of IL-12 therapy that elicit protective immune responses against the murine acute lymphoblastic leukaemia (ALL) cell line, 70Z/3. These systems differ in the method of IL-12 administration and ultimately result in leukaemia clearance by distinct mechanisms, emphasizing the importance of treatment vehicle. Injecting low-dose IL-12 was sufficient to elicit long-term protective immunity against an established leukaemia burden, mediated by both CD4(+) and CD8(+) T cells. These findings agree with the standard model of IL-12 activity. We compared this protocol to a cell-based approach in which a novel lentiviral vector (LV) expressing murine IL-12 was created, 70Z/3 cells transduced, and clones selected that stably secrete different amounts of IL-12. We found that only a small proportion (1%) of IL-12 secreting cells were required for rejection but that the amount of IL-12 produced per cell was critical for successful therapy. Importantly, the levels of IL-12 required were found to be higher than the levels reported to date in the human clinical trial literature. We found that the cell-based approach led to protective immunity that was both long-term and specific but dependent primarily on a CD4(+) cellular subset alone. Our results highlight that the mode of IL-12 delivery has a distinct impact on the immune response initiated, leading to leukaemia clearance by disparate mechanisms. We also establish a new and critical parameter, IL-12 production/cell, which may have significant implications for future therapeutic design.

A gain-of-function screen for genes that influence axon guidance identifies the NF-kappaB protein dorsal and reveals a requirement for the kinase Pelle in Drosophila photoreceptor axon targeting.

To identify novel regulators of nervous system development, we used the GAL4-UAS misexpression system in Drosophila to screen for genes that influence axon guidance in developing embryos. We mobilized the Gene Search (GS) P element and identified 42 lines with insertions in unique loci, including leak/roundabout2, which encodes an axon guidance receptor and confirms the utility of our screen. The genes we identified encode proteins of diverse classes, some acting near the cell surface and others in the cytoplasm or nucleus. We found that one GS line drove misexpression of the NF-kappaB transcription factor Dorsal, causing motor axons to bypass their correct termination sites. In the developing visual system, Dorsal misexpression also caused photoreceptor axons to reach incorrect positions within the optic lobe. This mistargeting occurred without observable changes of cell fate and correlated with localization of ectopic Dorsal in distal axons. We found that Dorsal and its inhibitor Cactus are expressed in photoreceptors, though neither was required for axon targeting. However, mutation analyses of genes known to act upstream of Dorsal revealed a requirement for the interleukin receptor-associated kinase family kinase Pelle for layer-specific targeting of photoreceptor axons, validating our screen as a means to identify new molecular determinants of nervous system development in vivo.

Longitudinal glia in the fly CNS: pushing the envelope on glial diversity and neuron-glial interactions.

Interactions between neurons and glial cells are crucial for nervous system development and function in all complex organisms, and many functional, morphological and molecular features of glia are well conserved among species. Here we review studies of the longitudinal glia (LG) in the Drosophila CNS. The LG envelop the neuropil in a membrane sheath, and have features resembling both oligodendrocytes and astrocytes. Because of their unique lineage, morphology and molecular features, the LG provide an excellent model to study the genetic mechanisms underlying glial subtype differentiation and diversity, glial morphogenesis and neuron-glial interactions during development. In addition, they are proving useful in understanding how glial cells maintain ion and neurotransmitter homeostasis and protect neurons from environmental insult.

Murine model of immune-mediated rejection of the acute lymphoblastic leukemia 70Z/3.

70Z/3 is a murine pre-B cell leukemia line derived from BDF(1) mice and has been used in the study of signaling pathways in B cells. 70Z/3 cells were initially found to cause widespread disease upon injections in animals. We have isolated 70Z/3 variants divergent in their capacity to lead to morbidity after injections. One variant, 70Z/3-NL, elicits an immune response protecting the animal from tumor growth. Another variant, 70Z/3-L, does not induce an effective immune response and causes morbidity. We demonstrated that both CD4(+) and CD8(+) T cells are required for the rejection of 70Z/3-NL cells. Interestingly, the immune response generated against 70Z/3-NL cells was found to protect against a challenge with the lethal variant, 70Z/3-L. This indicates that although both lines can be recognized and killed by the immune system, only 70Z/3-NL is capable of inducing a protective response. Further observations, using subclones isolated from 70Z/3-NL, demonstrated that immune recognition of a portion of the cells was sufficient for protection. Depletion of CD4(+) and CD8(+) T cells in animals injected previously with 70Z/3-NL cells showed that T cells, and not Abs, were required for the maintenance of the protection initiated by 70Z/3-NL. We tested the capacity of 70Z/3-NL cells to treat mice challenged with 70Z/3-L. We can delay injections of 70Z/3-NL and still provide protection for the animals. We have a model of immune-mediated rejection which will allow us to dissect the requirements for the initiation of immune responses against an ALL tumor cell line.