Role of aluminum-containing adjuvants in antigen internalization by dendritic cells in vitro

An important step in the induction of an immune response to vaccines is the internalization of antigens by antigen presenting cells, such as dendritic cells (DCs). Many current vaccines are formulated with antigens adsorbed to an aluminum-containing adjuvant. Following injection of the vaccine the antigens may either elute or stay adsorbed to the adjuvant surface. Antigens, which elute from the adjuvant surface, are internalized by dendritic cells through macropinocytosis while those that remain adsorbed are internalized with the adjuvant particle by phagocytosis. The relative efficiency of these two routes of internalization was studied. Alpha casein (AC) labeled with a green fluorescent dye was selected as the model antigen. In order to model vaccine antigens that elute from aluminum-containing adjuvants following administration, dendritic cells were incubated with a solution of fluorochrome-labeled alpha casein. To model vaccine antigens that do not elute from aluminum-containing adjuvants following administration, dendritic cells were exposed to fluorochrome-labeled alpha casein adsorbed to aluminum hydroxide adjuvant (AH). Alpha casein has eight phosphate groups and adsorbs to aluminum hydroxide adjuvant through ligand exchange. Alpha casein does not elute from aluminum hydroxide adjuvant upon exposure to cell culture media. The uptake of antigen by dendritic cells was determined at 0.5, 1, 2 and 3h by confocal microscopy and flow cytometry. Dendritic cells internalized both alpha casein in solution and alpha casein adsorbed to aluminum hydroxide adjuvant. However, the mean fluorescence intensity of dendritic cells incubated with adsorbed alpha casein was four times greater than dendritic cells incubated with alpha casein in solution. In addition, the internalization of alpha casein was enhanced when the mean aggregate diameter of the adjuvant in the cell culture media was reduced from 17 microm to 3 microm. It was concluded that antigen internalization by dendritic cells was enhanced when the antigen remained adsorbed to the aluminum-containing adjuvant following administration and the aggregate size of the adjuvant was smaller than dendritic cells which are approximately 10 microm in diameter.     

Putative role of nitric oxide synthase isoforms in the changes of nitric oxide concentration in rat brain cortex and cerebellum following sevoflurane and isoflurane anaesthesia

We have previously observed an increase in nitric oxide (NO) content in rat brain cortex following halothane, sevoflurane or isoflurane anaesthesia. This study was undertaken in order to determine whether isoform-specific nitric oxide synthase (NOS) inhibitors and inducers could modify these increases in NO contents. Rats were subjected to isoflurane and sevoflurane anaesthesia with concomitant administration of neuronal nitric oxide synthase (nNOS) inhibitor 7-Nitro-indazole (7-NI), inducible nitric oxide synthase (iNOS) inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT) or lipopolysaccharide. NO concentration in different organs was measured by electron paramagnetic resonance (EPR) spectroscopy. 7-NI significantly decreased NO concentration in cerebellum but not in brain cortex, whereas AMT decreased NO in all the organs studied. Anaesthesia significantly increased NO concentration in brain cortex and decreased that in cerebellum. AMT abolished the NO increase in brain cortex. Anaesthesia enhanced the drastic increase in NO concentration in brain cortex after intraventricular lipopolysaccharide administration. Isoflurane was found to inhibit recombinant nNOS and iNOS activities at high concentrations (EC50=20 mM). Our data suggest a putative role for iNOS in the increase in NO levels produced by isoflurane and sevoflurane, whereas nNOS activity is probably inhibited during anaesthesia.     

The advanced-DiaRem score improves prediction of diabetes remission 1 year post-Roux-en-Y gastric bypass

Aims/hypothesis

Not all people with type 2 diabetes who undergo bariatric surgery achieve diabetes remission. Thus it is critical to develop methods for predicting outcomes that are applicable for clinical practice. The DiaRem score is relevant for predicting diabetes remission post-Roux-en-Y gastric bypass (RYGB), but it is not accurate for all individuals across the entire spectrum of scores. We aimed to develop an improved scoring system for predicting diabetes remission following RYGB (the Advanced-DiaRem [Ad-DiaRem]).

Methods

We used a retrospective French cohort (n = 1866) that included 352 individuals with type 2 diabetes followed for 1 year post-RYGB. We developed the Ad-DiaRem in a test cohort (n = 213) and examined its accuracy in independent cohorts from France (n = 134) and Israel (n = 99).

Results

Adding two clinical variables (diabetes duration and number of glucose-lowering agents) to the original DiaRem and modifying the penalties for each category led to improved predictive performance for Ad-DiaRem. Ad-DiaRem displayed improved area under the receiver operating characteristic curve and predictive accuracy compared with DiaRem (0.911 vs 0.856 and 0.841 vs 0.789, respectively; p = 0.03); thus correcting classification for 8% of those initially misclassified with DiaRem. With Ad-DiaRem, there were also fewer misclassifications of individuals with mid-range scores. This improved predictive performance was confirmed in independent cohorts.

Conclusions/interpretation

We propose the Ad-DiaRem, which includes two additional clinical variables, as an optimised tool with improved accuracy to predict diabetes remission 1 year post-RYGB. This tool might be helpful for personalised management of individuals with diabetes when considering bariatric surgery in routine care, ultimately contributing to precision medicine.

     

Systemic mycophenolate mofetil avoids immune reactions in penetrating high-risk keratoplasty: preliminary results of an ongoing prospectively randomized multicentre study.

Recently, in a monocentre study mycophenolate mofetil (MMF) was demonstrated to be efficacious and safe in penetrating high-risk keratoplasty. Here, preliminary results of a randomized multicentre trial are presented. To date, 86 of 140 scheduled patients undergoing high-risk penetrating keratoplasty have already been randomized into the two study groups: 48 into the MMF group and 38 into the control group. All 86 patients received fluocortolon 1 mg/kg body weight/day, tapered within 3 weeks, and topical prednisolone acetate 1% tapered within 5 months. MMF was administered at a daily oral dose of 2 x 1000 mg for the first 6 postoperative months. Thereafter, MMF was tapered within 2 weeks. The proportion of grafts with immune reactions and side-effects were the main outcome measures. Within an average follow up of 9.2 +/- 6.6 months two patients developed reversible endothelial immune reactions in the MMF group after cessation of MMF application. In the control group, five reversible and three irreversible immune reactions were observed within an average follow up of 10.1 +/- 7.6 months. According to Kaplan and Meier analysis, the ratio of grafts without immune reactions was estimated 89% 1 year postoperatively in the MMF group, in contrast to only 67% in the control group (P = 0.03; log-rank test). Fifteen patients experienced side-effects, especially gastroenterotoxicity, tachycardia, arthralgia or systemic infections. All attributable side-effects were reversible. Systemic MMF may be an effective and safe immune modulating drug in the prophylaxis of immune reactions after penetrating high-risk keratoplasty.     

Induction of Antigen-Specific Th1-Type Immune Responses by Gamma-Irradiated Recombinant Brucella abortus RB51

Brucella abortus strain RB51 is an attenuated rough mutant used as the live vaccine against bovine brucellosis in the United States and other countries. We previously reported the development of strain RB51 as a bacterial vaccine vector for inducing Th1-type immune responses against heterologous proteins. Because safety concerns may preclude the use of strain RB51-based recombinant live vaccines, we explored the ability of a gamma-irradiated recombinant RB51 strain to induce heterologous antigen-specific immune responses in BALB/c mice. Exposure of strain RB51G/LacZ expressing Escherichia coli β-galactosidase to a minimum of 300 kilorads of gamma radiation resulted in complete loss of replicative ability. These bacteria, however, remained metabolically active and continued to synthesize β-galactosidase. A single intraperitoneal inoculation of mice with 10⁹ CFU equivalents of gamma-irradiated, but not heat-killed, RB51G/LacZ induced a β-galactosidase-specific Th1-type immune response. Though no obvious differences were detected in immune responses to B. abortus-specific antigens, mice vaccinated with gamma-irradiated, but not heat-killed, RB51G/LacZ developed significant protection against challenge with virulent B. abortus. In vitro experiments indicated that gamma-irradiated and heat-killed RB51G/LacZ induced maturation of dendritic cells; however, stimulation with gamma-irradiated bacteria resulted in more interleukin-12 secretion. These results suggest that recombinant RB51 strains exposed to an appropriate minimum dose of gamma radiation are unable to replicate but retain their ability to stimulate Th1 immune responses against the heterologous antigens and confer protection against B. abortus challenge in mice.     

Helicobacter pylori and allergy: Update of research

Recently a lot of literature has been published about the possible preventive action of Helicobacter pylori (H. pylori) against allergy. The present review summarizes research data about the association between H. pylori and allergic diseases, as well as discusses possible hypotheses about the preventive action of H. pylori against atopy. There is evidence from observational studies to support a weak inverse association between prevalence of H. pylori infection and allergy. However, confounders like some unidentified socioeconomic factors, antibiotic use and others could bias the association. Although data from cohort studies point to a possible association of H. pylori with some of the allergic diseases, no definite proof for causal relationship has been clearly demonstrated yet. A biological mechanism proposed to explain the preventive action of H. pylori to allergy is reduced exposure to a major stimulus for the generation of Treg cells in individuals without H. pylori infection. In addition, H. pylori could be an indicator for changes in gut microbiome, reflecting the complex interaction between microbes and immune system.     

Induction of antigen-specific Th1-type immune responses by gamma-irradiated recombinant Brucella abortus RB51

Brucella abortus strain RB51 is an attenuated rough mutant used as the live vaccine against bovine brucellosis in the United States and other countries. We previously reported the development of strain RB51 as a bacterial vaccine vector for inducing Th1-type immune responses against heterologous proteins. Because safety concerns may preclude the use of strain RB51-based recombinant live vaccines, we explored the ability of a gamma-irradiated recombinant RB51 strain to induce heterologous antigen-specific immune responses in BALB/c mice. Exposure of strain RB51G/LacZ expressing Escherichia coli beta-galactosidase to a minimum of 300 kilorads of gamma radiation resulted in complete loss of replicative ability. These bacteria, however, remained metabolically active and continued to synthesize beta-galactosidase. A single intraperitoneal inoculation of mice with 10(9) CFU equivalents of gamma-irradiated, but not heat-killed, RB51G/LacZ induced a beta-galactosidase-specific Th1-type immune response. Though no obvious differences were detected in immune responses to B. abortus-specific antigens, mice vaccinated with gamma-irradiated, but not heat-killed, RB51G/LacZ developed significant protection against challenge with virulent B. abortus. In vitro experiments indicated that gamma-irradiated and heat-killed RB51G/LacZ induced maturation of dendritic cells; however, stimulation with gamma-irradiated bacteria resulted in more interleukin-12 secretion. These results suggest that recombinant RB51 strains exposed to an appropriate minimum dose of gamma radiation are unable to replicate but retain their ability to stimulate Th1 immune responses against the heterologous antigens and confer protection against B. abortus challenge in mice.     

Follow up study of radiocesium contamination in a Greek forest ecosystem

Radiocesiun distribution in the different parts of a Quercus conferta Kit ecosystem in Northern Greece was measured in 2005-2006, twenty years after the Chernobyl accident. The comparison between the results of this study and those previously measured (1993-1995) in the same ecosystem gives information about the long-term behavior of 137Cs in forest ecosystems. The major part of the 137Cs inventory is still in the upper layers of the soil. The radiocesium distribution in soil is fixed and has been in equilibrium at least since 1993, when the first measurements were performed. The major contamination mechanism of leaves and wood is root uptake.     

Relationship between the strength of antigen adsorption to an aluminum-containing adjuvant and the immune response

Adsorption of the antigen to an aluminum-containing adjuvant is considered an important aspect of vaccine formulation. Adsorption is described by two parameters: the maximum amount that can be adsorbed as a monolayer, which is characterized by the adsorptive capacity and the strength of the adsorption force, which is described by the adsorptive coefficient. Research to date has focused on the adsorptive capacity with the goal of complete adsorption of the antigen. In this study, the relationship between the adsorptive coefficient and immunopotentiation was investigated. Four vaccines were prepared in which the adsorptive coefficient was varied by altering the number of phosphate groups on the antigen (alpha casein and dephosphorylated alpha casein) or the number of surface hydroxyls on the adjuvant (aluminum hydroxide adjuvant and phosphate-treated aluminum hydroxide adjuvant). In vitro elution upon exposure to interstitial fluid or normal human plasma was inversely related to the adsorptive coefficient. The geometric mean antibody titer in mice was also inversely related to the adsorptive coefficient. T-cell activation was not observed in mice that received the vaccine with the greatest adsorptive coefficient (alpha casein/aluminum hydroxide adjuvant). This suggests that antigen processing and presentation to T-cells is impaired when the antigen is adsorbed too strongly.     

Activation of dendritic cells and induction of CD4(+) T cell differentiation by aluminum-containing adjuvants

Aluminum-containing adjuvants are widely used in licensed human and veterinary vaccines. However, the mechanism by which these adjuvants enhance the immune response and predominantly stimulate a T(H)2 humoral immune response is not well understood. In this study, the effects of aluminum hydroxide and aluminum phosphate adjuvants on antigen presentation, expression of costimulatory molecules and cytokines by mouse dendritic cells (DCs) and the ability of DCs to induce T helper cell differentiation were investigated. Dendritic cells pulsed with ovalbumin (OVA) adsorbed to aluminum-containing adjuvants activated antigen-specific T cells more effectively than DCs pulsed with OVA alone. Aluminum hydroxide adjuvant had a significantly stronger effect than aluminum phosphate adjuvant. Both aluminum-containing adjuvants significantly increased the expression of CD86 on DCs but only aluminum hydroxide adjuvant also induced moderate expression of CD80. Aluminum-containing adjuvants stimulated the release of IL-1beta and IL-18 from DCs via caspase-1 activation. DCs incubated with LPS and OVA induced T(H)1 differentiation of na?ve CD4(+) T cells. In contrast, DCs incubated with aluminum/OVA activated CD4(+) T cells to secrete IL-4 and IL-5 as well as IFN-gamma. Addition of neutralizing anti-IL-1beta antibodies decreased IL-5 production and addition of anti-IL-18 antibodies decreased both IL-4 and IL-5 production. Inhibition of IL-1beta and IL-18 secretion by DCs via inhibition of caspase-1 also led to a marked decrease of IL-4 and IL-5 by CD4(+) T cells. These results indicate that aluminum-containing adjuvants activate DCs and influence their ability to direct T(H)1 and T(H)2 responses through the secretion of IL-1beta and IL-18.