Effects of long-term cryopreservation of PBMC on recovery of B cell subpopulations.

Cryopreservation of human peripheral blood mononuclear cells (PBMC) is used in many clinical and research applications to avoid direct and on-site analysis of samples. Storage of PBMC further allows prequalification of donor cells for routine laboratory methods involving the evaluation of immune responses. Previous studies reported changes in cellular composition and phenotype of PBMC following the freezing procedure. In our 12-month follow-up study, we focused on B cells and proportional representation of B cell subpopulations during long-term storage at -80 °C. Over the 12-month period, we observed a gradual decline in B cell viability and recovery. Notably, no changes in the proportional representation of human B cell subpopulations occurred in this period and the functional response elicited by antigen and TLR9 ligand CpG remained comparable to that observed after short-term storage for one month.

Does tetanus vaccination contribute to reduced severity of the COVID-19 infection?

We present the hypothesis to the scientific community actively designing clinical trials and recommending public health guidelines to control the pandemic that - "Tetanus vaccination may be contributing to reduced severity of the COVID-19 infection" - and urge further research to validate or invalidate the effectiveness of the tetanus toxoid vaccine against COVID-19. This hypothesis was revealed by an explainable artificial intelligence system unleashed on open public biomedical datasets. As a foundation for scientific rigor, we describe the data and the artificial intelligence system, document the provenance and methodology used to derive the hypothesis and also gather potentially relevant data/evidence from recent studies. We conclude that while correlations may not be reason for causation, correlations from multiple sources is more than a serendipitous coincidence that is worthy of further and deeper investigation.

Vaccination and Immune Checkpoint Inhibitors: Does Vaccination Increase the Risk of Immune-related Adverse Events? A Systematic Review of Literature.

INTRODUCTION: Immune checkpoint inhibitors (ICIs) have become part of cancer treatments. Their main side effects are immune-related adverse events (irAEs). So far, there has been no recommendation regarding routine vaccinations during ICIs treatment. Clinicians are aware of the risk of irAEs increases in this specific situation. The aim of this review of literature is to summarize the main studies about vaccination and ICIs interactions. METHODS: A systematic assessment of literature articles was performed by searching in PubMed (MEDLINE), and major oncology meeting following PRISMA guidelines. RESULTS: This review highlights the lack of literature. Indeed, most of the studies published were about influenza vaccination. Vaccination for patients under ICIs causes a humoral response and seems to be associated with an increase rate of seroconversion. Interestingly vaccination may provoke irAEs in ICIs-treated patients. So far, inactivated vaccines have not been contraindicated during ICI treatment. CONCLUSION: Larger prospective studies are needed in order to define a consensus on the use of vaccines under immunotherapy.

Generalized, non-neonatial tetanus is a highly fatal disease in Afghanistan: A case series study.

OBJECTIVES: To describe the clinical features and outcomes of a case series of adult tetanus and illustrate inadequacies in confronting this preventable disease. DESIGN AND METHODS: This study retrospectively evaluated 24 relatively severe, confirmed cases of tetanus, diagnosed between March 2017 and December 2018, in Kabul Antani Hospital, Afghanistan. RESULTS: Regarding the source of the infection: 18 patients (75%) had a history of injuries, 1 had a history of a dog bite and 1 was an intravenous drug user; 4 patients had no external injuries or wounds. Dysphagia was the main clinical manifestation for which patients sought medical treatment (50%). Of the 12 patients who died, 7 presented with confusion and seizure, 1 with acute kidney injury, and 2 with pneumonia. CONCLUSIONS: Mortality due to tetanus is high in Afghanistan (Case Fatality Rate (CFR) 50%)), suggesting an urgent need for vaccination policy and programs, post-exposure protocols, and facilities equipped for the treatment of adult tetanus. The Ministry of Public Health of Afghanistan should seek to improve the accessibility, distribution and recording of tetanus immunization through vaccination.

Effectiveness and selectivity of a heroin conjugate vaccine to attenuate heroin, 6-acetylmorphine, and morphine antinociception in rats: Comparison with naltrexone.

BACKGROUND: One emerging strategy to address the opioid crisis includes opioid-targeted immunopharmacotherapies. This study compared effectiveness of a heroin-tetanus toxoid (TT) conjugate vaccine to antagonize heroin, 6-acetylmorphine (6-AM), morphine, and fentanyl antinociception in rats. METHODS: Adult male and female Sprague Dawley rats received three doses of active or control vaccine at weeks 0, 2, and 4. Vaccine pharmacological selectivity was assessed by comparing opioid dose-effect curves in 50 °C warm-water tail-withdrawal procedure before and after active or control heroin-TT vaccine. Route of heroin administration [subcutaneous (SC) vs. intravenous [IV)] was also examined as a determinant of vaccine effectiveness. Continuous naltrexone treatment (0.0032-0.032 mg/kg/h) effects on heroin, 6-AM, and morphine antinociceptive potency were also determined as a benchmark for minimal vaccine effectiveness. RESULTS: The heroin-TT vaccine decreased potency of SC heroin (5-fold), IV heroin (3-fold), and IV 6-AM (3-fold) for several weeks without affecting IV morphine or SC and IV fentanyl potency. The control vaccine did not alter potency of any opioid. Naltrexone dose-dependently decreased antinociceptive potency of SC heroin, and treatment with 0.01 mg/kg/h naltrexone produced similar, approximate 8-fold decreases in potencies of SC and IV heroin, IV 6-AM, and IV morphine. The combination of naltrexone and active vaccine was more effective than naltrexone alone to antagonize SC heroin but not IV heroin. CONCLUSIONS: The heroin-TT vaccine formulation examined is less effective, but more selective, than chronic naltrexone to attenuate heroin antinociception in rats. Furthermore, these results provide an empirical framework for future preclinical opioid vaccine research to benchmark effectiveness against naltrexone.

Injectable Polypeptide Hydrogel Depot System for Assessment of the Immune Response-Inducing Efficacy of Sustained Antigen Release Alone.

Vaccines typically contain an antigen, delivery system (vehicle), and adjuvant, all of which contribute to inducing a potent immune response. Consequently, design of new vaccines is difficult, because the contributions and interactions of these components are difficult to distinguish. Here, it is aimed to develop an easy-to-use, non-immunogenic, injectable depot system for sustained antigen release that will be suitable for assessing the efficacy of prolonged antigen exposure per se for inducing an immune response. This should mimic real-life infections. Recombinant elastin-like polypeptides with periodic cysteine residues (cELPs) are selected, which reportedly show little or no immunogenicity, as carriers and tetanus toxoid (Ttd) as an antigen. After subcutaneous injection of the mixture, cELP rapidly forms a disulfide cross-linked hydrogel in situ, within which Ttd is physically incorporated, affording a biodegradable antigen depot. A series of Ttd-containing hydrogels is examined. A single injection induces high levels of tetanus antibody with high avidity for at least 20 weeks in mice. The chain length of cELP proves critical, whereas differences in hydrophobicity has little effect, although hydrophilic cELPs are more rapidly biodegraded. This system's ability to distinguish the contribution of sustained antigen release to antibody induction should be helpful for rational design of next-generation vaccines.

Vaccine blunts fentanyl potency in male rhesus monkeys.

One proposed factor contributing to the increased frequency of opioid overdose deaths is the emergence of novel synthetic opioids, including illicit fentanyl and fentanyl analogues. A treatment strategy currently under development to address the ongoing opioid crisis is immunopharmacotherapies or opioid-targeted vaccines. The present study determined the effectiveness and selectivity of a fentanyl-tetanus toxoid conjugate vaccine to alter the behavioral effects of fentanyl and a structurally dissimilar mu-opioid agonist oxycodone in male rhesus monkeys (n = 3-4). Fentanyl and oxycodone produced dose-dependent suppression of behavior in an assay of schedule-controlled responding and antinociception in an assay of thermal nociception (50 °C). Acute naltrexone (0.032 mg/kg) produced an approximate 10-fold potency shift for fentanyl to decrease operant responding. The fentanyl vaccine was administered at weeks 0, 2, 4, 9, 19, and 44 and fentanyl or oxycodone potencies in both behavioral assays were redetermined over the course of 49 weeks. The vaccine significantly and selectively shifted fentanyl potency at least 10-fold in both assays at several time points over the entire experimental period. Mid-point titer levels correlated with fentanyl antinociceptive potency shifts. Antibody affinity for fentanyl as measured by a competitive binding assay improved over time to approximately 3-4 nM. The fentanyl vaccine also increased fentanyl plasma levels approximately 6-fold consistent with the hypothesis that the vaccine sequesters fentanyl in the blood. Overall, these results support the continued development and evaluation of this fentanyl vaccine in humans to address the ongoing opioid crisis.

Preclinical efficacy of an anti-methamphetamine vaccine using E6020 adjuvant.

BACKGROUND AND OBJECTIVE: Methamphetamine (MA) substance use disorder (SUD) does not have an efficacious pharmacotherapy. We developed a MA vaccine and investigated its potential to attenuate MA induced responses. METHODS: We examined a novel adjuvant, E6020, a Toll-like receptor-4 (TLR-4) agonist combined with tetanus-toxoid conjugated to succinyl-methamphetamine (TT-SMA) adsorbed on aluminum hydroxide (alum). Adult BALB/c female mice received the vaccine and booster injections at weeks 0, 3, and 6. The efficacy of the vaccine was assessed by the level and affinity of anti-MA antibodies elicited, its ability to attenuate MA induced locomotor activation and its reduction in the amount of MA entering the brains of vaccinated mice. RESULTS: The TT-SMA vaccine containing alum and E6020 adjuvant produced anti-MA antibodies with nanomolar affinities and showed threefold greater peak titer levels than without E6020 (700 vs 250 µg/ml). These antibodies significantly decreased MA-induced locomotor activation (p < .05), and reduced the brain (p < .005) MA levels following MA administration in actively immunized mice. CONCLUSIONS: Thus, this anti-MA vaccine formulated with E6020 demonstrated effective functional protection against behavioral disruptions induced by MA. SCIENTIFIC SIGNIFICANCE: Together, anti-MA vaccine showing a promising improvement in the efficacy of the vaccine that could be an effective candidate vaccine for methamphetamine use disorder (MUD). Furthermore, combinations of adjuvants may be a tool to design vaccines for MA dependence in humans. (Am J Addict 2019;XX:1-8).

Synthesis of peptide based epsilon toxin vaccine by covalent anchoring to tetanus toxoid.

The epsilon toxin (Etx) produced by Clostridium perfringens type B and D causes severe enterotoxaemia associated with a general edema and neurological alterations, leading to subsequent death and is listed as one of the most lethal toxins. Currently employed vaccines against C. perfringens epsilon toxin include toxoid based vaccines. Use of peptide vaccines has become an interesting approach for vaccination after the successful licensing of peptide vaccines against Haemophilus influenza, Neisseria meningitides and Streptococcus pneumonia that have demonstrated the potential and effectiveness of these vaccines. Therefore, the present study was undertaken to develop a peptide based vaccine against epsilon toxin. Peptides were selected on the basis of epitope mapping by making 35 overlapping peptides of 15 amino acid residues in length specific to the primary amino acid sequence of the toxin, with a 7 amino acid residues overlaps between sequential peptides. Chemically synthesized peptides that were recognised by the antibody against the full length epsilon toxin were further assessed for vaccine potential. The selected peptides were chemically conjugated to partially reduced tetanus toxoid (TT) using of N-succinimidyl-3(2-pyridyldithio) propionate. Immunization of BALB/c mice with TT-peptide conjugates by sub-cutaneous route induced sustained high level mixed immune response as analyzed by antibody isotyping. Immunoblot analysis and ELISA clearly indicated generation of Etx-specific antibodies. Further, neutralization studies with the antisera generated against the TT-conjugated peptide(s) demonstrated that the antisera were able to neutralize the lethal dose of epsilon toxin in vitro demonstrating its potential as a promising vaccine candidate against enterotoxaemia.

Immunological evaluation of chitosan nanoparticles loaded with tetanus toxoid.

OBJECTIVES: The present study was aimed at comparing tetanus toxoid (TT)­loaded-chitosan nanoparticles with aluminum hydroxide as a common vaccine adjuvant. BACKGROUND: Tetanus remains to be a major public health problem. Nanoparticles have been extensively used as immune adjuvants. Tetanus toxoid (TT) encapsulated in chitosan nanoparticles is considered to be a promising tetanus vaccine candidate. METHODS: TT­loaded chitosan nanoparticles were prepared by the ionic gelation method. The nanoparticles were studied by SEM for their size and morphology. In vivo study was conducted to evaluate the immunity response using mice divided into 4 groups and injected with encapsulated toxoid. The immune responses were then measured using indirect ELISA. RESULTS: The purity and integrity of antigen were confirmed by SDS-PAGE electrophoresis. The size of nanoparticles was estimated at 100 nm. As a result, the IgG antibody levels were 1.9, 1.76, and 0.87 in chitosan nanoparticles, aluminum hydroxide, and TT alone groups, respectively. Also, the immune responses were significantly higher in immunized groups compared to control groups vaccinated with free adjuvant vaccines (p < 0.05). CONCLUSIONS: The quality and efficacy of toxoid­loaded chitosan nanoparticles were reasonable. It enhanced the immune responses as much as aluminum hydroxide adjuvant does and thus may be a good alternative candidate (Tab. 1, Fig. 3, Ref. 16).

Immune activation and HIV-specific T cell responses are modulated by a cyclooxygenase-2 inhibitor in untreated HIV-infected individuals: An exploratory clinical trial.

Pathologically elevated immune activation and inflammation contribute to HIV disease progression and immunodeficiency, potentially mediated by elevated levels of prostaglandin E2, which suppress HIV-specific T cell responses. We have previously shown that a high dose of the cyclooxygenase-2 inhibitor celecoxib can reduce HIV-associated immune activation and improve IgG responses to T cell-dependent vaccines. In this follow-up study, we included 56 HIV-infected adults, 28 antiretroviral therapy (ART)-naïve and 28 on ART with undetectable plasma viremia but CD4 counts below 500 cells/µL. Patients in each of the two study groups were randomized to receive 90 mg qd of the cyclooxygenase-2 inhibitor etoricoxib for six months, two weeks or to a control arm, respectively. T cell activation status, HIV Gag-specific T cell responses and plasma inflammatory markers, tryptophan metabolism and thrombin generation were analyzed at baseline and after four months. In addition, patients received tetanus toxoid, conjugated pneumococcal and seasonal influenza vaccines, to which IgG responses were determined after four weeks. In ART-naïve patients, etoricoxib reduced the density of the activation marker CD38 in multiple CD8+ T cell subsets, improved Gag-specific T cell responses, and reduced in vitro plasma thrombin generation, while no effects were seen on plasma markers of inflammation or tryptophan metabolism. No significant immunological effects of etoricoxib were observed in ART-treated patients. Patients receiving long-term etoricoxib treatment had poorer tetanus toxoid and conjugated pneumococcal vaccine responses than those receiving short-course etoricoxib. Cyclooxygenase-2 inhibitors may attenuate harmful immune activation in HIV-infected patients without access to ART.

The Effect of ASP2409, a Novel CD86-Selective Variant of CTLA4-Ig, on Renal Allograft Rejection in Nonhuman Primates.

BACKGROUND: Blockade of CD28-mediated T cell costimulation by a modified cytotoxic T lymphocyte-associated antigen 4 (CTLA4-Ig), belatacept, is a clinically effective immunosuppressive therapy for the prevention of renal allograft rejection. Use of belatacept-based calcineurin inhibitor-free immunosuppression, however, has demonstrated an increased frequency of cellular rejection episodes and immunosuppression-related safety issues relative to conventional regimens. Furthermore, belatacept typically requires infusion for its administration chronically, which may present an inconvenience to patients. To address these issues, a novel CTLA4-Ig variant, ASP2409, with improved CD86 binding selectivity and affinity relative to belatacept was created using DNA shuffling directed evolution methods. METHODS: We evaluated the immunosuppressive effect of ASP2409 on in vitro alloimmune T cell responses, in vivo tetanus toxoid (TTx)-induced immunological responses and renal transplantation in cynomolgus monkeys. RESULTS: ASP2409 had 6.1-fold higher and 2.1-fold lower binding affinity to monkey CD86 and CD80 relative to belatacept, respectively. ASP2409 was 18-fold more potent in suppressing in vitro alloimmune T cell responses relative to belatacept. In a cynomolgus monkey TTx immunization model, ASP2409 inhibited anti-TTx immune responses at a 10-fold lower dose level than belatacept. In a cynomolgus monkey renal transplantation model, subcutaneous injection of 1 mg/kg ASP2409 prevented allograft rejection through complete CD86 and partial CD80 receptor occupancies and dramatically prolonged renal allograft survival in combination with tacrolimus or mycophenolate mofetil/methylprednisolone. CONCLUSIONS: These results support the potential of ASP2409 as an improved CTLA4-Ig for maintenance immunosuppression in organ transplantation.

Novel Sperm and Gonadotropin-releasing Hormone-based Recombinant Fusion Protein: Achievement of 100% Contraceptive Efficacy by Co-immunization of Male and Female Mice.

Improvements in long-term female contraception can be achieved by vaccinating with sperm-derived proteins. Here, recombinant proteins comprising either (i) N- (amino acid residues 1-80) or C- (amino acid residues 76-126) terminal fragments of mouse sperm protein 17 (Sp17) fused to the promiscuous T non-B cell epitope of tetanus toxoid (TT), amino acid residues 830-844 followed by di-lysine linker (KK) (TT-KK-Sp17N or TT-KK-Sp17C , respectively) or (ii) mouse equatorin (amino acid residues 21-185) fused to the T non-B cell epitope of bovine RNase (amino acid residues 94-104) were expressed in Escherichia coli. Immunization of female FVB/J mice, using alum as an adjuvant, led to the generation of high antibody titers against the above proteins. Antibodies against both N- and C-terminal fragments of Sp17 reacted with the entire capacitated mouse spermatozoa, whereas those against equatorin reacted exclusively with the equatorial region. Despite the reactivity of all immune sera, only sera from mice immunized with TT-KK-Sp17N and TT-KK-Sp17C significantly reduced mouse in vitro fertilization. Mating studies of the immunized females with un-immunized male mice revealed the highest infertility in the TT-KK-Sp17C -immunized group. In an attempt to further boost the immune response, the C-terminal fragment of Sp17 was expressed as fusion protein with a tandem repeat of gonadotropin-releasing hormone (GnRH) (Sp17C -GnRH2 ). Immunization of both male and female mice with Sp17C -GnRH2 led to higher contraceptive efficacy compared to mice immunized with TT-KK-Sp17C . Interestingly, mating studies wherein partners were both immunized with Sp17C -GnRH2 showed a complete failure of female mice to conceive. Thus, immunization of both males and females with Sp17C -GnRH2 has the potential to increase contraceptive efficacy. Mol. Reprod. Dev. 83: 1048-1059, 2016. © 2016 Wiley Periodicals, Inc.

Tetanus toxoid-loaded cationic non-aggregated nanostructured lipid particles triggered strong humoral and cellular immune responses.

In the present investigation, non-aggregated cationic and unmodified nanoparticles (TT-C-NLPs4 and TT-NLPs1) were prepared of about 49.2 ± 6.8-nm and 40.8 ± 8.3-nm, respectively. In addition, spherical shape, crystalline architecture and cationic charge were also noticed. Furthermore, integrity and conformational stability of TT were maintained in both TT-C-NLPs4 and TT-NLPs1, as evidenced by symmetrical position of bands and superimposed spectra, respectively in SDS-PAGE and circular dichroism. Cellular uptake in RAW264.7 cells indicating the concentration-dependent internalisation of nanoparticles. Qualitatively, CLSM exhibited enhanced cellular uptake of non-aggregated TT-C-NLPs4 owing to interaction with negatively charged plasma membrane and clevaloe mediated/independent endocytosis. In last, in vivo immunisation with non-aggregated TT-C-NLPs4 elicited strong humoral (anti-TT IgG) and cellular (IFN-γ) immune responses at day 42, as compared to non-aggregated TT-NLPs1 and TT-Alum following booster immunisation at day 14 and 28. Thus, non-aggregated cationic lipid nanoparticles may be a potent immune-adjuvant for parenteral delivery of weak antigens.

Maternal colostral leukocytes appear to enhance cell-mediated recall response, but inhibit humoral recall response in prime-boost vaccinated calves.

Whether colostral leukocytes (CLs) of vaccinated dams influence the immune response of neonatal calves following vaccination against the same antigen as their respective dams remains unanswered. Therefore, we compared the induction of humoral and cellular immune responses after vaccination in calves that had received CL-free or maternal CL-enriched colostrum from a cell-free colostrum bank of nonvaccinated cows. Also, vaccinated calves that had received fresh maternal colostrum from their own dam were included in the study. Moreover, we analyzed whether the post-partum time of priming vaccination (day 2, 5 or 10) of the calves could influence the outcome of the immune responses. All calves received a booster vaccination 23 days after the priming vaccination. All calves showed only an increase in tetanus toxoid (TT)-specific antibodies and TT-induced proliferation after booster vaccination. Tetanus toxoid-specific antibody responses in calves increased immediately after booster vaccination, irrespective of whether or not their cell-free bank colostrum had been enriched with CLs from their own dam. Conversely, calves receiving their own plain dam colostrum displayed a later humoral response, due to colostral antibodies. After booster vaccination, calves of the CL-enriched colostrum group had a more pronounced antigen-specific proliferative response than the calves of the CL-free colostrum group. We propose that CLs might have a suppressive influence on the emergence of the TT-specific antibodies, but an enhancing effect on the TT-specific lymphocyte proliferation of newborn calves upon TT vaccination, which is dependent on the time point of the priming vaccination.

Altered methamphetamine place conditioning in mice vaccinated with a succinyl-methamphetamine-tetanus-toxoid vaccine.

BACKGROUND AND OBJECTIVES: We previously reported that an anti-methamphetamine (MA) vaccine attenuated drug-conditioned effects in mice, but it used a carrier protein and adjuvant not available for clinical use. Here we produced a vaccine with the same hapten (succinyl-methamphetamine, SMA) but attached to tetanus toxoid (SMA-TT) and adsorbed to aluminum hydroxide, components approved for use in humans. We then assessed the vaccine's ability to generate anti-MA antibodies, alter acquisition and reinstatement of MA place conditioning, and prevent MA brain penetration. METHODS: Mice were administered SMA-TT at weeks 0 and 3 and non-vaccinated mice received saline. Anti-MA antibody concentrations were determined at 8 and 12 weeks. Place conditioning began during week 9 in which vaccinated and non-vaccinated mice were divided into groups and conditioned with .5, or 2.0 mg/kg MA. Following acquisition training, mice were extinguished and then a reinstatement test was performed in which mice were administered their original training dose of MA. Separate groups of non-vaccinated and vaccinated mice were administered .5 and 2.0 mg/kg MA and brain MA levels determined. RESULTS AND CONCLUSIONS: Anti-MA antibody levels were elevated at week 8 and remained so through week 12. The SMA-TT vaccine attenuated acquisition and reinstatement of MA place conditioning. Significantly greater proportions of vaccinated mice during acquisition and reinstatement tests showed conditioned place aversion. Moreover, MA brain levels were decreased in vaccinated mice following administration of both doses of MA. SCIENTIFIC SIGNIFICANCE: Results support further development of anti-MA vaccines using components approved for use in humans.

Poor functional immune recovery in aged HIV-1-infected patients following successfully treatment with antiretroviral therapy.

Aging is now a well-recognized characteristic of the HIV-infected population and both AIDS and aging are characterized by a deficiency of the T-cell compartment. The objective of the present study was to evaluate the impact of antiretroviral (ARV) therapy in recovering functional response of T cells to both HIV-1-specific ENV peptides (ENV) and tetanus toxoid (TT), in young and aged AIDS patients who responded to ARV therapy by controlling virus replication and elevating CD4(+) T cell counts. Here, we observed that proliferative response of T-cells to either HIV-1-specific Env peptides or tetanus toxoid (TT) was significantly lower in older antiretroviral (ARV)-treated patients. With regard to cytokine profile, lower levels of IFN-γ, IL-17 and IL-21, associated with elevated IL-10 release, were produced by Env- or TT-stimulated T-cells from older patients. The IL-10 neutralization by anti-IL-10 mAb did not elevate IFN-γ and IL-21 release in older patients. Finally, even after a booster dose of TT, reduced anti-TT IgG titers were quantified in older AIDS patients and it was related to both lower IL-21 and IFN-γ production and reduced frequency of central memory T-cells. Our results reveal that ARV therapy, despite the adequate recovery of CD4(+) T cell counts and suppression of viremia, was less efficient in recovering adequate immune response in older AIDS patients.

Dietary exposure to benzoxazinoids enhances bacteria-induced monokine responses by peripheral blood mononuclear cells.

SCOPE: To examine potentially immunomodulating effects of dietary benzoxazinoids (BXs), present in cereal grains. METHODS AND RESULTS: Nineteen healthy volunteers were randomly distributed into two groups, who received diets with high or low content of BXs for 3 wk. After a week's wash-out, the groups switched diets. Peripheral blood mononuclear cells (PBMCs) were stimulated with Porphyromonas gingivalis, Escherichia coli lipopolysaccharide (LPS), or tetanus toxoid (TT). PBMCs from a healthy donor received the same stimuli in presence of serum from each participant receiving BXs. The production of monokines, T-cell cytokines and T-helper cell proliferation were assessed. A 3-wk diet with high BX content enhanced IL-1ß responses against LPS and P. gingivalis, as well as TNF-α response against P. gingivalis, after 24 h of stimulation. Moreover, IL-6 was found to be increased after 7 days of stimulation with LPS. No effect was observed on T-cell cytokines or proliferation. BX levels in serum after a single meal did not modify cytokine responses. CONCLUSION: High dietary intake of BXs enhances bacteria-induced production of pro-inflammatory monokines by PBMCs, but not T-cell responses; presumably due to intrinsic changes within PBMCs, built up over 3 wk of BX-rich diet, rather than to an immediate effects of BXs contained in serum.

Total synthesis of tetraacylated phosphatidylinositol hexamannoside and evaluation of its immunomodulatory activity.

Tuberculosis, aggravated by drug-resistant strains and HIV co-infection of the causative agent Mycobacterium tuberculosis, is a global problem that affects millions of people. With essential immunoregulatory roles, phosphatidylinositol mannosides are among the cell-envelope components critical to the pathogenesis and survival of M. tuberculosis inside its host. Here we report the first synthesis of the highly complex tetraacylated phosphatidylinositol hexamannoside (Ac2PIM6), having stearic and tuberculostearic acids as lipid components. Our effort makes use of stereoelectronic and steric effects to control the regioselective and stereoselective outcomes and minimize the synthetic steps, particularly in the key desymmetrization and functionalization of myo-inositol. A short synthesis of tuberculostearic acid in six steps from the Roche ester is also described. Mice exposed to the synthesized Ac2PIM6 exhibit increased production of interleukin-4 and interferon-γ, and the corresponding adjuvant effect is shown by the induction of ovalbumin- and tetanus toxoid-specific antibodies.