Sex-Related Effect of Aging in Gingival Gamma-Delta T Cells.

Aging affects the number and function of gamma-delta (γδ) T cells in a tissue-specific manner, modifying the risk for inflammatory disease. These aging-related γδT-cell variations in gingival tissues that could increase the risk for inflammation and periodontal disease remain unknown. Here we sought to identify quantitative and qualitative variations in gingival γδT cells associated with aging that could have an impact in oral immunoinflammatory responses. For this, gingival tissues from young (4 mo) and aged (24 mo) male and female mice were collected and analyzed by flow cytometry. Cell suspensions were stimulated and stained with eFluor450 (cell viability), anti-CD45 (hematopoietic cells), anti-CD3 (lymphocytes), anti-TCRγδ (γδT cells), anti-IL-15rα (cell proliferation), and anti-Notch-3 (senescence marker). Detection of intracellular cytokines IL-17A and interferon γ (IFNγ) was performed. Gingival expression of specific γ- and δ-chains and cytokines was evaluated by quantitative reverse transcription polymerase chain reaction. A significantly higher number of IL-17A-producing γδT cells and IL-17A expression levels were observed in gingival tissues from aged females but not males. Similarly, the number of gingival Notch-3+ γδT cells increased with aging only in females. IL-15rα was not detected in gingival γδT cells. Chains γ1, 2, 4, 5, 6, and 7 as well as δ1, 2, 4, and 6 were detected. Detection levels of all γ chains except γ1 as well as δ1 and δ2 changed with aging in males, females, or both. Interestingly, number of IL-17A-producing conventional T cells similarly increased with aging only in females. Both sexes showed increased IFNγ+ conventional T-cell numbers with aging; however, it reached significance only in females. In conclusion, the number of gingival IL-17A-producing γδT cells and IL-17A expression increase naturally with aging specifically in females. This sexual dimorphism in gingival γδT and conventional Th17 cell numbers and phenotypes suggests distinct aging-related mechanisms of periodontitis in males and females.

The contribution of sublingual immunotherapy to the achievement of control in birch-related mild persistent asthma: A real-life randomised trial

Background: Asthma control represents the main goal of asthma management and different strategies aim to avoid the long term downsides of inhaled corticosteroids. We investigated in real-life conditions the contribution of sublingual immunotherapy in achieving the control of birch-related mild persistent asthma compared to two usual step-up therapeutic options. Methods: A three-year open randomised study included 84 asthmatics, uncontrolled during the previous birch pollen season, despite a treatment with budesonide 400 μg/day. Patients randomly received budesonide 800 μg/day, budesonide 1600 μg/day, budesonide 400 μg/day plus montelukast 10 μg/day and budesonide 400 μg/day plus carbamylated allergoid of betulaceae pre-coseasonally. Asthma Control test, combined allergy symptoms and medications score, albuterol consumption, lung function, nasal eosinophils and nasal steroids usage were assessed as changes from the first to last pollen season. Result: Seventy-six patients concluded the study. All options, except budesonide 800 μg/day, produced an improvement of mean monthly Asthma Control test (p < 0.05). Patients undergoing low-dose budesonide plus immunotherapy achieved, after three years, an appreciable control (ACT mean score 24). A significant improvement was seen in all groups for allergy symptoms plus medications and bronchial reactivity. Albuterol consumption and lung function improved in all but the first group. Only budesonide plus immunotherapy reduced nasal eosinophils and nasal steroids usage. Two mild self-resolving adverse events were reported. Conclusions: For patients with respiratory allergy due to birch pollen and mild persistent asthma, sublingual immunotherapy added to low-dose inhaled corticosteroids appears effective in maintaining long-term seasonal asthma control, representing a safe opportunity to reduce the cumulative amount of delivered corticosteroids (AU)

The contribution of sublingual immunotherapy to the achievement of control in birch-related mild persistent asthma: a real-life randomised trial.

BACKGROUND: Asthma control represents the main goal of asthma management and different strategies aim to avoid the long term downsides of inhaled corticosteroids. We investigated in real-life conditions the contribution of sublingual immunotherapy in achieving the control of birch-related mild persistent asthma compared to two usual step-up therapeutic options. METHODS: A three-year open randomised study included 84 asthmatics, uncontrolled during the previous birch pollen season, despite a treatment with budesonide 400µg/day. Patients randomly received budesonide 800µg/day, budesonide 1600µg/day, budesonide 400µg/day plus montelukast 10µg/day and budesonide 400µg/day plus carbamylated allergoid of betulaceae pre-coseasonally. Asthma Control test, combined allergy symptoms and medications score, albuterol consumption, lung function, nasal eosinophils and nasal steroids usage were assessed as changes from the first to last pollen season. RESULT: Seventy-six patients concluded the study. All options, except budesonide 800µg/day, produced an improvement of mean monthly Asthma Control test (p<0.05). Patients undergoing low-dose budesonide plus immunotherapy achieved, after three years, an appreciable control (ACT mean score 24). A significant improvement was seen in all groups for allergy symptoms plus medications and bronchial reactivity. Albuterol consumption and lung function improved in all but the first group. Only budesonide plus immunotherapy reduced nasal eosinophils and nasal steroids usage. Two mild self-resolving adverse events were reported. CONCLUSIONS: For patients with respiratory allergy due to birch pollen and mild persistent asthma, sublingual immunotherapy added to low-dose inhaled corticosteroids appears effective in maintaining long-term seasonal asthma control, representing a safe opportunity to reduce the cumulative amount of delivered corticosteroids.

Effect of two doses of carbamylated allergoid extract of dust mite on nasal reactivity.

Background and Objective. Single SLIT studies with native allergen extracts support a dose-response effect for clinical and immunological outcomes. Conversely for carbamylated allergoids this dose-response effects is less evident, likely because the threshold for efficacy is more easily reached through the enhanced bioavailability of the extract consequent to the selective chemical modification. Thus this pilot study investigates the dose-response effect on nasal specific reactivity and safety of two unusual doses of carbamylated allergoid in patients mono-sensitized to house dust mites. Methods. A prospective open randomized study involved 6-65 year-old Italian patients with clinically relevant sensitization to house dust mites and positive response to nasal provocation challenge. Monomeric carbamylated allergoid was delivered once daily at the dose of 1000 AU or 2000 AU from June to September 2009, during the lowest level of mites exposure. Primary outcomes were the change of the threshold of allergen concentration for a positive nasal provocation test (NPT) before and after the treatment and the product safety. Secondary outcome was the change  in the mean percentage fall of peak nasal inspiratory flow (PNIF) following nasal challenge. Results. Thirty-four patients were enrolled. Fifteen in group 1 and 14 in group 2 concluded the study. After 12 weeks all patients treated in group 1 and all but one in group 2 showed an increase in the threshold dose provoking a positive NPT. Those with no symptoms onset with the highest dose delivered were 80% in group 1 and 78.6% in group 2 (p=0.92). From first to second challenge, the mean percentage fall of PNIF  was reduced with no statistical difference between groups (p=0.95), and with no difference between the final mean percentage falls (p=0.65). No serious adverse reactions occurred and the frequency of events, all mild, was similar in the two groups. Conclusions. Twelve weeks of carbamylated sublingual allergoid delivered at 1000AU or 2000AU once daily appear equally safe and show comparable effect in increasing  the threshold of allergen concentration for a positive nasal provocation test, confirming the apparent absence of a dose response effect for the used doses.

Targeted deletion of MyD88 in intestinal epithelial cells results in compromised antibacterial immunity associated with downregulation of polymeric immunoglobulin receptor, mucin-2, and antibacterial peptides.

Intestinal epithelial cells (IECs) form a physical and immunological barrier that separates the vast gut microbiota from host tissues. MyD88-dependent Toll-like receptor signaling is a key mediator of microbial-host cross-talk. We examined the role of epithelial MyD88 expression by generating mice with an IEC-targeted deletion of the Myd88 gene (MyD88(ΔIEC)). Loss of epithelial MyD88 signaling resulted in increased numbers of mucus-associated bacteria; translocation of bacteria, including the opportunistic pathogen Klebsiella pneumoniae, to mesenteric lymph nodes; reduced transmucosal electrical resistance; impaired mucus-associated antimicrobial activity; and downregulated expression of polymeric immunoglobulin receptor (the epithelial IgA transporter), mucin-2 (the major protein of intestinal mucus), and the antimicrobial peptides RegIIIγ and Defa-rs1. We further observed significant differences in the composition of the gut microbiota between MyD88(ΔIEC) mice and wild-type littermates. These physical, immunological, and microbial defects resulted in increased susceptibility of MyD88(ΔIEC) mice to experimental colitis. We conclude that MyD88 signaling in IECs is crucial for maintenance of gut homeostasis.

Regulation of the polymeric immunoglobulin receptor by the classical and alternative NF-κB pathways in intestinal epithelial cells.

The polymeric immunoglobulin receptor (pIgR) transports IgA antibodies across intestinal epithelial cells (IECs). Expression of pIgR is upregulated by proinflammatory signaling pathways via activation of nuclear factor-κB (NF-κB). Here, we examined the contributions of the RelA-dependent classical and RelB-dependent alternative pathways of NF-κB to pIgR regulation in the HT-29 human IEC line following stimulation with tumor necrosis factor (TNF), lipopolysaccharide (LPS; Toll-like receptor 4 (TLR4) ligand), and polyinosinic: polycytidylic acid (pIC; TLR3 ligand). Whereas induction of proinflammatory genes such as interleukin-8 (IL-8) required only RelA, pIgR expression was regulated by complex mechanisms that involved both RelA and RelB. Upregulation of pIgR expression by ligation of the lymphotoxin-ß receptor suggested a direct role for the alternative NF-κB pathway. Inhibition of mitogen-activated protein kinases reduced the induction of IL-8, but enhanced the induction of pIgR by TNF and TLR signaling. Regulation of pIgR through unique signaling pathways could allow IECs to sustain high levels of IgA transport while limiting the proinflammatory responses.

Efficacy and safety of sublingual immunotherapy with grass monomeric allergoid: comparison between two different treatment regimens.

BACKGROUND: Sublingual immunotherapy (SLIT) with monomeric carbamylated allergoid proved to be well tolerated, safe and effective in patients with respiratory allergy. Standard administration regimens are expected to require a long time before clinical benefit can be appreciated. We investigated whether pre-seasonal and perennial regimens differently affect the clinical efficacy of grass pollen SLIT. METHODS: Adult patients with allergic rhino-conjunctivitis with/without mild intermittent asthma due to grass pollen were included into this open prospective study and randomised to receive SLIT with a continuous regimen (Group 1: 1,000 AU/week for the entire study period) or a pre-seasonal regimen (Group 2: 5,000 AU/week for 10 weeks/year for 2 years), or on demand drug therapy alone (Group 3) for two years. At entry (November 2005), at the end of the first and second pollen season, a Visual Analogue Scale (VAS) was used to assess patients' well-being. Symptom score and drug consumption were evaluated during the seasons. Methacholine challenge was performed at study entry and conclusion. Adverse events were recorded along the whole study duration. RESULTS: Thirty-two patients were divided into Group 1 (n = 10), Group 2 (n = 11) and Group 3 (n = 11). A significant VAS improvement was observed in both SLIT groups, after the first and second pollen season, compared to baseline and to Group 3 (p < 0.05). Less symptoms and need for medications resulted during the second season (p < 0.05). No relevant variations in bronchial hyper-reactivity have been observed between the three groups. Only 2 patients experienced local or mild reactions in SLIT groups. CONCLUSION: Both pre-seasonal and continuous regimen of SLIT with monomeric allergoid turned out effective and safe, suggesting that a pre-seasonal course with 5,000 AU/week for 10 weeks may represent a convenient option in patients with grass pollen allergic rhinitis with/without mild intermittent asthma. Further research is urgently needed to consolidate these preliminary evidences.

Sublingual allergoid immunotherapy: a new 4-day induction phase in patients allergic to house dust mites.

Sublingual immunotherapy with monomeric allergoid (allergoid SLIT), given according to the standard scheme, has proved effective and safe in many clinical trials. However, its build-up phase requires a long time ranging from 16 days to 14 weeks. This study therefore investigated whether, with a four-day up-dosing, the same benefit could be achieved in a shorter time. Thirty rhinitic and/or asthmatic patients (16 M and 14 F, mean age 36+/-8.2 years) allergic to house dust mites (HDM) with or without other sensitizations were randomized to allergoid SLIT or standard drug therapy. The build-up phase lasted four days. The first day the patients took a 300 AU tablet, the second day two 300 AU tablets, the third day three 300 AU tablets and the fourth day four 300 AU tablets. The total amount taken during the up-dosing was 3000 AU. Patients were then treated for 12 months at the dosage of 2000 AU/week (total amount of allergen: 104,000 AU/year). The symptom score and drug consumption were recorded from November to February on monthly diary cards. At baseline and after 12 months a Visual Analogue Scale (VAS) was used to rate the patients? well-being. Skin prick test reactivity was evaluated before and after the 12-month treatment in both groups using 10 mg/mL histamine as reference. VAS scores rose significantly (about 45%) in both groups in comparison to baseline (p=0.001). In addition, there was a significantly greater reduction of the global symptoms score (about 52%) - but not in drug consumption - in the SLIT group in comparison to controls (p=0.0004). The SLIT group showed a highly significant reduction (about 39%) in skin prick test reactivity (p=0.000003) while the control group remained unchanged (p=0.5226). No severe adverse events were observed. Even with this short four-day up-dosing, the allergoid SLIT proves to be safe. In addition, it is already effective in patients allergic to HDM after 12 months, and significantly reduces allergen-specific skin reactivity.

Oral hyposensitization to nickel induces clinical improvement and a decrease in TH1 and TH2 cytokines in patients with systemic nickel allergy syndrome.

Some patients with nickel (Ni) allergic contact dermatitis suffer from systemic (intestinal or cutaneous) symptoms after ingestion of Ni-rich foods and experience symptoms reduction with low-Ni diet, a condition termed Systemic Ni Allergy Syndrome (SNAS). We aimed at evaluating whether oral administration of low nickel doses improved clinical conditions and modulated immunological aspects of SNAS, without significant side effects. Thirty-six SNAS patients were enrolled. Treatment started after 1-month of low-Ni diet and consisted in an incremental oral NiOH dose phase (0.3ng to 1.5 microg/week) followed by a 12-months maintenance phase (1.5 microg/week). Randomly, twenty-four patients added Ni therapy to low-Ni diet and 12 remained with diet alone. All patients were allowed rescue medications (antihistamines and topical steroids). After 4 months, Ni-rich foods were gradually reintroduced. In vitro allergen-driven IL13, IL5 and IFN-gamma release by peripheral blood mononuclear cells was evaluated before and after treatment. Twenty-three patients receiving NiOH and the 12 control patients completed the study. Evaluation of SNAS clinical severity (by VAS and drug consumption) showed a significant difference in favor of NiOH-treated patients compared to controls. Twenty of 23 patients in the NiOH group and none in the control group tolerated Ni-rich food reintroduction. Release of all studied cytokines in culture supernatants was significantly lower after NiOH treatment. In conclusion NiOH is effective in reducing symptoms and drug consumption in SNAS and is able to modulate inflammatory parameters.

Efficacy, safety and tolerability of sublingual monomeric allergoid in tablets given without up-dosing to pediatric patients with allergic rhinitis and/or asthma due to grass pollen.

The efficacy and safety of monomeric allergoid (Lofarma, Milan) have been demonstrated in adults but very few studies have examined it in children. This study therefore investigated the efficacy and safety of this sublingual immunotherapy (SLIT) at the dosage of 1000 AU five times a week without any up-dosing. Forty allergic children (17 M and 23 F, mean age 7 years, range 4-16 years), 16 with rhinitis and 24 with rhinitis and asthma, were randomized to SLIT or drug therapy. All the patients were sensitized to grass; some were also sensitized, though to a lesser extent, to Parietaria, Olea and Betulaceae. The patients were treated pre-/co-seasonally for two years. A visual analogue scale (VAS) was used at baseline and at the end of the first and second pollen seasons to rate the patients' well-being. The VAS score was significantly higher after both the first and the second year of treatment in the SLIT group than in the controls (p<0.05). It improved in comparison to baseline only in the active group. All 40 children tolerated the therapy very well. The monomeric allergoid at the dosage of 5000 AU/week thus appears to have a good efficacy and safety profile in children.

Signature biomarkers in Crohn's disease: toward a molecular classification.

In an effort to develop a molecular classification scheme for Crohn's disease (CD), mucosal biopsies from 69 CD patients and 28 normal controls were analyzed for expression of the RelA subunit of nuclear factor (NF)-kappaB, A20 (a negative regulator of NF-kappaB), polymeric immunoglobulin receptor (pIgR), tumor necrosis factor (TNF), and interleukin (IL)-8. Principal component analysis was used to classify individuals into three subsets based on patterns of biomarker expression. Set 1 included normal subjects and CD patients with mild disease and good responses to therapy, thus defining "normal" biomarker expression. CD patients in set 2, characterized by low expression of all five biomarkers, had moderate to severe disease and poor responses to immunosuppressive and anti-TNF therapy. Patients in set 3, characterized by low expression of RelA, A20, and pIgR, normal TNF and elevated IL-8, had acute inflammation that responded well to therapy. Classification of CD patients by these biomarkers may predict disease behavior and responses to therapy.

Sublingual immunotherapy for allergic respiratory disease in elderly patients: a retrospective study.

BACKGROUND: Very few studies have evaluated the effects of sublingual immunotherapy (SLIT) in elderly adults with either rhinitis or bronchial asthma. The aim of this study was to ascertain whether SLIT is effective in these patients. METHODS: One hundred and sixty seven patients (aged 18-65 years) with persistent rhinitis and mild asthma, selected from 573 subjects allergic to house-dust mites, were treated with either standard chronic pharmacotherapy or SLIT plus drugs on demand. Monthly symptom/drug scores, respiratory function, methacholine (MCh) challenge and eosinophil count were scheduled at the beginning and end of the study. RESULTS: We analysed two age groups (18-28 years, 49 patients) and 55-65 years, 40 patients). There were no differences between the groups at baseline but MCh sensitivity was lower in the older patients. At the end of treatment, SLIT achieved improvement in all variables (p< 0.001) in both age groups, but the global symptoms were lower in the younger patients (p=0.0002). There were also fewer new sensitizations in the SLIT groups (p=0.03) than in the "control"patients given standard pharmacotherapy, but with no relation to age. Asthma became worse only in the control groups, regardless of age. CONCLUSIONS: SLIT reduces symptoms, drug consumption and the progression of the disease in both young and elderly subjects allergic to house-dust mites, with persistent rhinitis and mild bronchial asthma.

Parietaria sublingual allergoid immunotherapy with a co-seasonal treatment schedule.

BACKGROUND: Sublingual immunotherapy (SLIT) with monomeric allergoid, given according to the standard scheme, resulted effective and safe. However, the achievement of a clinical benefit requires a long time. We thus performed this study using an administration protocol starting in the co-seasonal period with a 3-day build-up phase and lasting only 6 months, in order to obtain the above benefit in a shorter time. METHODS AND RESULTS: The study, prospective, randomised and controlled versus drug therapy, was conducted on 65 rhinitic and/or asthmatic patients allergic to Parietaria with or without other sensitisations. Twenty-four were allocated to 1,000 AU/week, 21 to 3,000 AU/week and 21 to drug therapy. They were treated from April to September 2006. At baseline, 3 and 6 months a Visual Analogue Scale (VAS) was performed to assess the patients' well-being. Drug consumption was evaluated by means of monthly diary cards. Bronchial reactivity was investigated at baseline and 6 months by methacholine challenge test. There was a greater VAS improvement in both the SLIT groups than in the controls after 6 months (p<0.05). In patients taking 3,000 AU/week this was already evident after 3 months. There was a significant reduction in rescue medication consumption between 3 and 6 months (p<0.05) in all three groups. The bronchial reactivity was reduced only in the SLIT groups (p<0.001). No adverse events were observed. CONCLUSIONS: At 6 months the allergoid SLIT showed itself to be effective and safe. In addition the subjective clinical benefit was obtained in a more rapid period, i.e. 3 instead of 6 months, when a higher maintenance dose was administered.

Safety, tolerability and efficacy of sublingual allergoid immunotherapy with a 4-day shortened build-up phase.

BACKGROUND: Sublingual specific immunotherapy (SLIT) with monomeric allergoid has shown to be safe and effective the studies performed so far. The build-up phase, however, is rather time consuming mainly if performed with the conventional schedule of 14 weeks. AIMS OF STUDY: We evaluated the possibility of shortening and simplifying this phase, through a new build-up scheme of only 4 days, as well as the persistence of the allergoid SLIT efficacy after 12 months. METHODS: Thirty-nine patients (26 M, 13 F, mean age 20.5 years, range 6-49) with a history of moderate/severe rhinitis with or without mild asthma due to perennial and/or seasonal allergens entered the study. The posological schedule, adopting only 1,000 AU tablets, was the following: 1/2 tablet the 1st day; 1/2 table twice the second day; 1/2 table plus 1 table the 3rd day, 1 tablet twice the 4th day; 1 tablet twice weekly from the 5th to the 365th day (maintenance therapy). RESULTS: Only two mild adverse reactions occurred during the initial phase which disappeared with the prosecution of the treatment. During the maintenance therapy no adverse event was observed. Symptoms improved consistently and drug consumption was reduced in most of the patients. CONCLUSIONS: The 4-day shortened build-up phase resulted to be safe, well tolerated and effective, already after one year of treatment.

Clinical efficacy and safety of preseasonal sublingual immunotherapy with grass pollen carbamylated allergoid in rhinitic patients. A double-blind, placebo-controlled study.

BACKGROUND: The aim of this study was to confirm the clinical efficacy and safety of a preseasonal sublingual immunotherapy (SLIT) in a group of allergic patients with seasonal rhinoconjunctivitis with or without mild intermittent or mild persistent asthma. The immunotherapy was administered through the oral mucosa with a monomeric carbamylated allergoid (allergoid SLIT) for grass pollens. A secondary endpoint was to evaluate the effect of the allergoid SLIT on nasal reactivity. METHODS AND RESULTS: A single-center, randomized, double-blind, placebo-controlled study was performed. Patients were selected and randomly allocated to two groups: one group received active treatment (allergoid SLIT) for 2 years and the other received placebo. Both groups received the necessary drug treatment throughout the trial. Thirty-three outpatients (20 men and 13 women, mean age: 30 years; range: 19-43) attending our center were enrolled in the study. Symptoms and medications were scored on diary cards during the pollen season. An allergen nasal challenge was performed at baseline and after 2 years of SLIT to evaluate nasal reactivity. Because the clinical scores were non-normally distributed, the Mann-Whitney and the Chi-square tests for intergroup comparisons and the Wilcoxon test for intragroup comparisons were used. The results were evaluated after 1 and 2 years of treatment. Between the first and second years of treatment, no changes in the scores for the placebo group were found, while for the active vaccine group significant decreases were found in rhinorrhea (p < 0.03), sneezing (p < 0.03), and conjunctivitis (p < 0.02). Symptom scores after nasal challenge decreased (p < 0.03) after 2 years' treatment. Nasal steroid use significantly decreased in the active treatment group during May and June in both the years of treatment (p < 0.02). Only two mild local adverse events were reported in the active group and none was reported in the placebo group. CONCLUSIONS: The results of this study show that the allergoid SLIT is safe and effective in decreasing symptom scores and drug use in rhinitic patients allergic to grass pollen.

Regulation of yeast CTP synthetase activity by protein kinase C.

CTP synthetase (EC 6.3.4.2, UTP:ammonia ligase (ADP-forming)) is an allosterically regulated enzyme in the yeast Saccharomyces cerevisiae. In this work we examined the regulation of CTP synthetase activity by S. cerevisiae protein kinase C (Pkc1p) phosphorylation. The results of labeling experiments with S. cerevisiae mutants expressing different levels of the PKC1 gene indicated that phosphorylation of CTP synthetase was mediated by Pkc1p in vivo. In vitro, Pkc1p phosphorylated purified CTP synthetase on serine and threonine residues, which resulted in the activation (3-fold) of enzyme activity. The mechanism of this activation involved an increase in the apparent Vmax of the reaction and an increase in the enzyme's affinity for ATP. In vitro phosphorylated CTP synthetase also exhibited a decrease in its positive cooperative kinetic behavior with respect to UTP and ATP. Phosphorylation of CTP synthetase did not have a significant effect on the kinetic properties of the enzyme with respect to glutamine and GTP. Phosphorylation of CTP synthetase resulted in a decrease in the enzyme's sensitivity to product inhibition by CTP. Phosphorylation did not affect the mechanism by which CTP inhibits CTP synthetase activity.

Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by CTP.

In the yeast Saccharomyces cerevisiae, the major membrane phospholipid phosphatidylcholine is synthesized by the CDP-diacylglycerol and CDP-choline pathways. We examined the regulation of phosphatidylcholine synthesis by CTP. The cellular concentration of CTP was elevated (2.4-fold) by overexpressing CTP synthetase, the enzyme responsible for the synthesis of CTP. The overexpression of CTP synthetase resulted in a 2-fold increase in the utilization of the CDP-choline pathway for phosphatidylcholine synthesis. The increase in CDP-choline pathway usage was not due to an increase in the expression of any of the enzymes in this pathway. CDP-choline, the product of the phosphocholine cytidylyltransferase reaction, was the limiting intermediate in the CDP-choline pathway. The apparent Km of CTP (1.4 mM) for phosphocholine cytidylyltransferase was 2-fold higher than the cellular concentration of CTP (0.7 mM) in control cells. This provided an explanation of why the overexpression of CTP synthetase caused an increase in the cellular concentration of CDP-choline. Phosphatidylserine synthase activity was reduced in cells overexpressing CTP synthetase. This was not due to a transcriptional repression mechanism. Instead, the decrease in phosphatidylserine synthase activity was due, at least in part, to a direct inhibition of activity by CTP. These results show that CTP plays a role in the regulation of the pathways by which phosphatidylcholine is synthesized. This regulation includes the supple of CTP for the phosphocholine cytidylyltransferase reaction in the CDP-choline pathway and the inhibition of the phosphatidylserine synthase reaction in the CDP-diacylglycerol pathway.

Evidence that dissipation of proton motive force is a common mechanism of action for bacteriocins and other antimicrobial proteins.

While bacteriocins from lactic acid bacteria (LAB) have generated tremendous interest among food microbiologists, they are not unique. The biosphere is awash with antimicrobial proteins such as colicins, defensins, cecropins, and magainins. These proteins share many characteristics. They are low molecular weight, cationic, amphiphilic, tend to aggregate and are benign to the producing organism. In cases where the mode of action has been investigated, the cell membrane appears to be the site of action. There is increasing evidence that bacteriocins from many bacterial genera also share these characteristics. After a brief introduction on the significance of LAB bacteriocins, this review provides some background on proton motive force. Current studies of mechanisms for various bacteriocins are reviewed. Evidence is then introduced that bacteriocins produced by lactic acid bacteria act by the common mechanism of depleting proton motive force. The role and importance of energized membranes in this process is examined. These observations are linked to literature which demonstrates that many other classes of antimicrobial proteins act by the same mechanism. Questions regarding the role of receptor proteins and the physical mechanism by which PMF is depleted remain unresolved.

Correlation of bioenergetic parameters with cell death in Listeria monocytogenes cells exposed to nisin.

In Listeria monocytogenes, nisin induced ATP efflux, reduced the intracellular ATP concentration within 1 min, and dissipated the proton motive force within 2 min. Efflux accounted for only 20% of the ATP depletion, suggesting that ATP hydrolysis also occurred. ATP efflux depended on nisin concentration and followed saturation kinetics. These results suggest that nisin breaches the membrane permeability barrier in a manner more consistent with pore formation than with a nonspecific detergent-like membrane destabilization.

Common mechanistic action of bacteriocins from lactic Acid bacteria.

The influence of four bacteriocins from lactic acid bacteria on the proton motive force (PMF) of sensitive organisms was investigated. Pediocin PA-1 (20 mug/ml) and leuconocin S (48.5 mug/ml) mediated total or major PMF dissipation of energized Listeria monocytogenes Scott A cells in a concentration-dependent manner, as has been shown for nisin. Lactacin F (13.5 mug/ml) caused 87% PMF depletion of energized Lactobacillus delbrueckii ATCC 4797 cells, also in a concentration-dependent fashion. The energy requirements for the activity of these four bacteriocins were determined by using the ionophores nigericin and valinomycin to carry out partial and specific deenergization of the target organisms. Pediocin PA-1, leuconocin S, and lactacin F acted in an energy-independent manner, whereas the activity of nisin was confirmed to be energy dependent. These results together with published reports on other bacteriocins suggest that the bacteriocins of lactic acid bacteria share a common mechanism, the depletion of PMF.