Short Vi-polysaccharide abrogates T-independent immune response and hyporesponsiveness elicited by long Vi-CRM197 conjugate vaccine.

Polysaccharide-protein conjugates have been developed to overcome the T-independent response, hyporesponsiveness to repeated vaccination, and poor immunogenicity in infants of polysaccharides. To address the impact of polysaccharide length, typhoid conjugates made with short- and long-chain fractions of Vi polysaccharide with average sizes of 9.5, 22.8, 42.7, 82.0, and 165 kDa were compared. Long-chain-conjugated Vi (165 kDa) induced a response in both wild-type and T cell-deficient mice, suggesting that it maintains a T-independent response. In marked contrast, short-chain Vi (9.5 to 42.7 kDa) conjugates induced a response in wild-type mice but not in T cell-deficient mice, suggesting that the response is dependent on T cell help. Mechanistically, this was explained in neonatal mice, in which long-chain, but not short-chain, Vi conjugate induced late apoptosis of Vi-specific B cells in spleen and early depletion of Vi-specific B cells in bone marrow, resulting in hyporesponsiveness and lack of long-term persistence of Vi-specific IgG in serum and IgG+ antibody-secreting cells in bone marrow. We conclude that while conjugation of long-chain Vi generates T-dependent antigens, the conjugates also retain T-independent properties, leading to detrimental effects on immune responses. The data reported here may explain some inconsistencies observed in clinical trials and help guide the design of effective conjugate vaccines.

Protection by vaccination of children against typhoid fever with a Vi-tetanus toxoid conjugate vaccine in urban Bangladesh: a cluster-randomised trial.

BACKGROUND: Typhoid fever remains a major cause of morbidity and mortality in low-income and middle-income countries. Vi-tetanus toxoid conjugate vaccine (Vi-TT) is recommended by WHO for implementation in high-burden countries, but there is little evidence about its ability to protect against clinical typhoid in such settings. METHODS: We did a participant-masked and observer-masked cluster-randomised trial preceded by a safety pilot phase in an urban endemic setting in Dhaka, Bangladesh. 150 clusters, each with approximately 1350 residents, were randomly assigned (1:1) to either Vi-TT or SA 14-14-2 Japanese encephalitis (JE) vaccine. Children aged 9 months to less than 16 years were invited via parent or guardian to receive a single, parenteral dose of vaccine according to their cluster of residence. The study population was followed for an average of 17·1 months. Total and overall protection by Vi-TT against blood culture-confirmed typhoid were the primary endpoints assessed in the intention-to-treat population of vaccinees or all residents in the clusters. A subset of approximately 4800 participants was assessed with active surveillance for adverse events. The trial is registered at www.isrctn.com, ISRCTN11643110. FINDINGS: 41 344 children were vaccinated in April-May, 2018, with another 20 412 children vaccinated at catch-up vaccination campaigns between September and December, 2018, and April and May, 2019. The incidence of typhoid fever (cases per 100 000 person-years) was 635 in JE vaccinees and 96 in Vi-TT vaccinees (total Vi-TT protection 85%; 97·5% CI 76 to 91, p<0·0001). Total vaccine protection was consistent in different age groups, including children vaccinated at ages under 2 years (81%; 95% CI 39 to 94, p=0·0052). The incidence was 213 among all residents in the JE clusters and 93 in the Vi-TT clusters (overall Vi-TT protection 57%; 97·5% CI 43 to 68, p<0·0001). We did not observe significant indirect vaccine protection by Vi-TT (19%; 95% CI -12 to 41, p=0·20). The vaccines were well tolerated, and no serious adverse events judged to be vaccine-related were observed. INTERPRETATION: Vi-TT provided protection against typhoid fever to children vaccinated between 9 months and less than 16 years. Longer-term follow-up will be needed to assess the duration of protection and the need for booster doses. FUNDING: The study was funded by the Bill & Melinda Gates Foundation.

Saccharide dosage content of meningococcal polysaccharide conjugate vaccines determined using WHO International Standards for serogroup A, C, W, Y and X polysaccharides.

Potency of meningococcal polysaccharide-protein conjugate vaccines relies on the polysaccharide content to prevent meningitis. NIBSC, as the official national control laboratory in UK, analysed ten different mono- and multi-meningococcal conjugate vaccines, using established International Standards for meningococcal serogroups A, C, W, Y and X, by resorcinol or HPAEC-PAD assay. Most saccharide contents were within ±20% of their claimed content for licensure with taking different O-acetylation levels into consideration, with only MenC content in two vaccines below (by 60% and 54%) the labelled value, however, previous study showed different dosage was not necessarily correlated to the immunogenicity of those vaccines. This study demonstrated the use of International Standards to quantify saccharide content in polysaccharide-based vaccines with different percentage of O-acetylation. These International Standards are suitable to serve as either quantitative standard or calibrator of in-house standards, with supplied stability data.

Novel Gellan Gum-Based In Situ Nanovesicle Formulation of Docetaxel for Its Localized Delivery Using Depot Formation.

In the present study, different in situ hydrogel formulations of docetaxel (DTX) based on biocompatible polymers such as Hyaluronic Acid (HA), poloxamer-407, chitosan and gellan gum were formulated to increase its therapeutic efficacy and reduce toxicity. DTX was loaded in nanovesicles (20 mg/mL equivalent to commercial strength) and further incorporated into the hydrogel bases to possess a dual rationale of protection against burst release and enhanced solubility of the drug. The optimized hydrogel formulation (NV-TPGS-3-GG-4) showed ideal rheological behavior and in situ characteristics at 37±0.5°C with sustained release of more than 144 h. The optimized formulation had instant in vitro gelation (2.8±0.3 min) with good injectability in comparison to the conventional commercial DTX injectable formulation having instant release (<2 h). Additionally, developed formulation exhibited an improved biodisponibility (25.1±0.2 h) in comparison to the commercially available formulation (1.7±0.1 h). The Solid Tumor Carcinoma model in Swiss albino mice revealed that the optimized formulation (based on gellan gum) showed better tumor reduction (85.7±1.2%) and lower toxicity as compared to the commercial formulation (77.3±1.3%). Pharmacokinetic and biodistribution studies demonstrated 3 to 4 times higher localization of drug in tumors. Our findings suggested that injectable gellan gum-based in situ hydrogel formulation can be an effective delivery system for DTX with enhanced solubility, reduced toxicity, and better targeting to the tumors for improved therapeutics.Graphical abstract.

Formulation development and in-vitro evaluation of gastroretentive drug delivery system of loxoprofen sodium: A natural excipients based approach.

The limitations of conventional type delivery systems to retain drug (s) in the stomach has resulted in the development of novel gastroretentive drug delivery system. We developed single-layer effervescent floating tablets of loxoprofen sodium for prolong delivery in the stomach using natural polymers xanthan gum, guar gum and semisynthetic polymer HPMCK4M. All the formulations (F1-F9) were developed by varying concentrations of xanthan gum and HPMCK4M while guar gum concentration was kept constant. Two gas generating agent (s) incorporated were sodium bicarbonate and citric acid. All compendial pre and post-compression tests results were in the acceptable limits. FTIR analysis confirmed drug-polymer compatibility. The in-vitro drug release in simulated conditions i.e., 0.1 N HCl for 12 h revealed orderly increase in total floating time, i.e., less than 6 h for F1 over 12 h for F9. Formulations F1 to F4 were not capable to retard drug release up to 12 h, whereas F5-F7 for 12 h, while F8 and F9 for more than 12 h. Data fitting in various kinetic models showed that drug release best fit in first order kinetic model and F9 in zero order. Based on results data, F7 was the best among all.

Comparative Study of Immunogenic Properties of Purified Capsular Polysaccharides from Streptococcus suis Serotypes 3, 7, 8, and 9: the Serotype 3 Polysaccharide Induces an Opsonizing IgG Response.

Streptococcus suis is an encapsulated bacterium and one of the most important swine pathogens and a zoonotic agent for which no effective vaccine exists. Bacterial capsular polysaccharides (CPSs) are poorly immunogenic, but anti-CPS antibodies are essential to the host defense against encapsulated bacteria. In addition to the previously known serotypes 2 and 14, which are nonimmunogenic, we have recently purified and described the CPS structures for serotypes 1, 1/2, 3, 7, 8, and 9. Here, we aimed to elucidate how these new structurally diverse CPSs interact with the immune system to generate anti-CPS antibody responses. CPS-stimulated dendritic cells produced significant levels of C-C motif chemokine ligand 3 (CCL3), partially via Toll-like receptor 2 (TLR2)- and myeloid differentiation factor 88-dependent pathways, and CCL2, via TLR-independent mechanisms. Mice immunized with purified serotype 3 CPS adjuvanted with TiterMax Gold produced an opsonizing IgG response, whereas other CPSs or adjuvants were negative. Mice hyperimmunized with heat-killed S. suis serotypes 3 and 9 both produced anti-CPS type 1 IgGs, whereas serotypes 7 and 8 remained negative. Also, mice infected with sublethal doses of S. suis serotype 3 produced primary anti-CPS IgM and IgG responses, of which only IgM were boosted after a secondary infection. In contrast, mice sublethally infected with S. suis serotype 9 produced weak anti-CPS IgM and IgG responses following a secondary infection. This study provides important information on the divergent evolution of CPS serotypes with highly different structural and/or biochemical properties within S. suis and their interaction with the immune system.

Suppression of Staphylococcus aureus Superantigen-Independent Interferon Gamma Response by a Probiotic Polysaccharide.

Staphylococcus aureus is a Gram-positive opportunistic pathogen that causes a variety of diseases. Bloodstream infection is the most severe, with mortality rates reaching 20 to 50%. Exopolysaccharide (EPS) from the probiotic Bacillus subtilis reduces bacterial burden and inflammation during S. aureus bloodstream infection in mice. Protection is due, in part, to hybrid macrophages that restrict S. aureus growth through reactive oxygen species and to limiting superantigen-induced T cell activation and interferon gamma (IFN-γ) production during infection. A decrease in IFN-γ production was observed within 24 h after infection, and here, we investigated how EPS abrogates its production. We discovered that S. aureus uses a rapid, superantigen-independent mechanism to induce host IFN-γ and that this is mediated by interleukin-12 (IL-12) activation of NK cells. Furthermore, we found that EPS limits IFN-γ production by modulating host immunity in a Toll-like receptor 4 (TLR4)-dependent manner, a signaling pathway that is required for EPS-mediated protection from S. aureus infection in vivo We conclude that EPS protects hosts from acute bloodstream S. aureus infection not only by inducing macrophages that restrict S. aureus growth and inhibit superantigen-activated T cells but also by limiting NK cell production of IFN-γ after S. aureus infection in a TLR4-dependent manner.

Effect of the live oral attenuated typhoid vaccine, Ty21a, on systemic and terminal ileum mucosal CD4+ T memory responses in humans.

Our current understanding of CD4+ T-cell-mediated immunity (CMI) elicited by the oral live attenuated typhoid vaccine Ty21a is primarily derived from studies using peripheral blood. Very limited data are available in humans regarding mucosal immunity (especially CD4+ T) at the site of infection (e.g. terminal ileum; TI). Here using multiparametric flow cytometry, we examined the effect of Ty21a immunization on TI-lamina propria mononuclear cells (LPMC) and peripheral blood CD4+ T memory (TM) subsets in volunteers undergoing routine colonoscopy. Interestingly, we observed significant increases in the frequencies of LPMC CD4+ T cells following Ty21a immunization, restricted to the T effector/memory (TEM)-CD45RA+ (TEMRA) subset. Importantly, Ty21a immunization elicited Salmonella Typhi-responsive LPMC CD4+ T cells in all major TM subsets [interferon (IFN)γ and interleukin (IL)-17A in TEM; IFNγ and macrophage inflammatory protein (MIP)1ß in T central/memory (TCM); and IL-2 in TEMRA]. Subsequently, we analyzed LPMC S. Typhi-responsive CD4+ T cells in depth for multifunctional (MF) effectors. We found that LPMC CD4+ TEM responses were mostly MF, except for those cells exhibiting the characteristics associated with IL-17A responses. Finally, we compared mucosal to systemic responses and observed that LPMC CD4+S. Typhi-specific responses were unique and distinct from their systemic counterparts. This study provides the first demonstration of S. Typhi-specific CD4+ TM responses in the human TI mucosa and provides valuable information about the generation of mucosal immune responses following oral Ty21a immunization.

Comprehensive mapping of the cell response to E. coli infection in porcine intestinal epithelial cells pretreated with exopolysaccharide derived from Lactobacillus reuteri.

Bacterial exopolysaccharides (EPSs) are known to modulate immunity. To date, a plethora of studies have reported the effect of EPSs on intestinal cells; however few works have revealed a complete picture of the signalling events in intestinal epithelial cells induced by bacterial EPSs. Here, using transcriptomics, we comprehensively mapped the biological processes in porcine intestinal epithelial cells challenged with EPS derived from Lactobacillus reuteri alone, enterotoxigenic Escherichia coli (ETEC) or ETEC after pretreatment with EPS. The Gene Ontology analysis of differentially expressed genes (DEGs) showed that ETEC is able to evoke biological processes specifically involved in cell junction reorganization, extracellular matrix degradation, and activation of the innate immune response through the activation of pattern recognition receptors, such as TLRs and CTRs. A total of 495 DEGs were induced in ETEC-challenged cells. On the other hand, EPS pretreatment was able to attenuate overexpression of the genes induced by ETEC infection. The most relevant finding of this study is that EPS has a suppressive effect on the inflammatory response evoked by ETEC infection. On the basis of high-throughput RNA-seq, this report is the first to describe the effects of EPSs derived from L. reuteri used as a pretreatment of global gene expression in porcine epithelial cells.

Differential effects of BCG vaccine on immune responses induced by vi polysaccharide typhoid fever vaccination: an explorative randomized trial.

The Vi polysaccharide typhoid fever vaccine (TFV) provides incomplete protection against typhoid fever. BCG, the vaccine against tuberculosis, can potentiate immune responses to other vaccines through induction of trained innate immunity and heterologous adaptive immunity. We performed an explorative, randomized, noncontrolled open trial to investigate whether BCG vaccination increases humoral and cellular response to TFV and whether BCG and TFV modulate nonspecific immune responses. Thirty volunteers were randomized to receive either TFV alone or BCG followed by TFV after 2 weeks. Ex vivo leukocyte responses and anti-Vi IgG antibody titers were measured 2 weeks and 3 months after TFV. BCG administration prior to TFV vaccination did not increase specific humoral or cellular immune responses to Salmonella typhi. TFV vaccination decreased pro-inflammatory responses to non-related stimuli. This effect was counteracted by prior BCG administration, which also led to decreased IL-10 and increased IL-22 responses to non-related stimuli. In an in vitro model of trained immunity TFV led to immunotolerance, which was partially reversed by BCG-induced trained immunity. BCG does not modulate adaptive immune responses to TFV but partially prevents inhibition of innate immune responses induced by TFV. Nonspecific effects of vaccines to unrelated microbial stimuli must be considered in the evaluation of their biological effects (ClinicalTrials.gov NCT02175420).

Broadly protective semi-synthetic glycoconjugate vaccine against pathogens capable of producing poly-ß-(1→6)-N-acetyl-d-glucosamine exopolysaccharide.

Poly-ß-(1→6)-N-acetylglucosamine (PNAG) was first discovered as a major component of biofilms formed by Staphylococcus aureus and some other staphylococci but later this exopolysaccharide was also found to be produced by pathogens of various nature. This common antigen is considered as a promising target for construction of a broadly protective vaccine. Extensive studies of PNAG, its de-N-acetylated derivative (dPNAG, containing around 15% of residual N-acetates) and their conjugates with Tetanus Toxoid (TT) revealed the crucial role of de-N-acetylated glucosamine units for the induction of protective immunity. Conjugates of synthetic penta- (5GlcNH2) and nona-ß-(1→6)-d-glucosamines (9GlcNH2) were tested in vitro and in different animal models and proved to be effective in passive and active protection against different microbial pathogens. Presently conjugate 5GlcNH2-TT is being produced under GMP conditions and undergoes safety and effectiveness evaluation in humans and economically important animals. Current review summarizes all stages of this long-termed study.

Structure-based glycoconjugate vaccine design: The example of Group B Streptococcus type III capsular polysaccharide.

Microbial surface polysaccharides are important virulence factors and targets for vaccine development. Glycoconjugate vaccines, obtained by covalently linking carbohydrates and proteins, are well established tools for prevention of bacterial infections. Elucidation of the minimal portion involved in the interactions with functional antibodies is of utmost importance for the understanding of their mechanism of induction of protective immune responses and the design of synthetic glycan based vaccines. Typically, this is achieved by combination of different techniques, which include ELISA, glycoarray, Surface Plasmon Resonance in conjunction with approaches for mapping at atomic level the position involved in binding, such as Saturation Transfer NMR and X-ray crystallography. This review provides an overview of the structural studies performed to map glycan epitopes (glycotopes), with focus on the highly complex structure of Group B Streptococcus type III (GBSIII) capsular polysaccharide. Furthermore, it describes the rational process followed to translate the obtained information into the design of a protective glycoconjugate vaccine based on a well-defined synthetic glycan epitope.

Design and development of spirulina polysaccharide-loaded nanoemulsions with improved the antitumor effects of paclitaxel.

Aims: In this study, we prepared spirulina polysaccharides into spirulina polysaccharide-loaded nanoemulsions (SPS-NEs), and determined the antitumor effect of SPS-NEs, when combined with paclitaxel (PTX).Methods: SPS-NEs were prepared by a phase transformation method. The Characterisation and stability of SPS-NEs was measured. The antitumor effect of SPS-NEs combined with PTX was determined by S180 cells or RAW 264.7 macrophages and S180 tumour-bearing mice.Results: SPS-NEs were spherical and stable, the particle size of SPS-NEs was 84.6 ± 3.31 nm, PDI = 0.235 ± 0.02. PTX + SPS-NEs exhibited a much greater toxicity against RAW 264.7 cells than PTX. PTX + SPS-NEs increased the release of NO, IL-6 and TNF-α, and the expression of p-p65 NF-κB, p-I-κB, TLR4. In addition, PTX + SPS-NEs significantly inhibited tumour growth by 72.82% and increased the secretion of serum IL-2, TNF-α and IFN-γ.Conclusions: SPS-NEs can regulate immunity through TLR4/NF-κB signalling pathways, which enhances the anti-tumour effect of PTX.

Mini-GAGR, an intranasally applied polysaccharide, activates the neuronal Nrf2-mediated antioxidant defense system.

Oxidative stress triggers and exacerbates neurodegeneration in Alzheimer's disease (AD). Various antioxidants reduce oxidative stress, but these agents have little efficacy due to poor blood-brain barrier (BBB) permeability. Additionally, single-modal antioxidants are easily overwhelmed by global oxidative stress. Activating nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2) and its downstream antioxidant system are considered very effective for reducing global oxidative stress. Thus far, only a few BBB-permeable agents activate the Nrf2-dependent antioxidant system. Here, we discovered a BBB-bypassing Nrf2-activating polysaccharide that may attenuate AD pathogenesis. Mini-GAGR, a 0.7-kDa cleavage product of low-acyl gellan gum, increased the levels and activities of Nrf2-dependent antioxidant enzymes, decreased reactive oxygen species (ROS) under oxidative stress in mouse cortical neurons, and robustly protected mitochondria from oxidative insults. Moreover, mini-GAGR increased the nuclear localization and transcriptional activity of Nrf2 similarly to known Nrf2 activators. Mechanistically, mini-GAGR increased the dissociation of Nrf2 from its inhibitor, Kelch-like ECH-associated protein 1 (Keap1), and induced phosphorylation and nuclear translocation of Nrf2 in a protein kinase C (PKC)- and fibroblast growth factor receptor (FGFR1)-dependent manner. Finally, 20-day intranasal treatment of 3xTg-AD mice with 100 nmol of mini-GAGR increased nuclear p-Nrf2 and growth-associated protein 43 (GAP43) levels in hippocampal neurons, reduced p-tau and ß-amyloid (Aß) peptide-stained neurons, and improved memory. The BBB-bypassing Nrf2-activating polysaccharide reported here may be effective in reducing oxidative stress and neurodegeneration in AD.

Probiotic Exopolysaccharide Protects against Systemic Staphylococcus aureus Infection, Inducing Dual-Functioning Macrophages That Restrict Bacterial Growth and Limit Inflammation.

Staphylococcus aureus causes severe systemic infection with high mortality rates. We previously identified exopolysaccharide (EPS) from a probiotic, Bacillus subtilis, that induces anti-inflammatory macrophages with an M2 phenotype and protects mice from Citrobacter rodentium-induced colitis. We tested if EPS could protect from systemic infection induced by S. aureus and found that EPS-treated mice had enhanced survival as well as reduced weight loss, systemic inflammation, and bacterial burden. While macrophages from EPS-treated mice display an M2 phenotype, they also restrict growth of internalized S. aureus through reactive oxygen species (ROS), reminiscent of proinflammatory phagocytes. These EPS-induced macrophages also limit T cell activation by S. aureus superantigens, and EPS abrogates systemic induction of gamma interferon after infection. We conclude that B. subtilis EPS is an immunomodulatory agent that induces hybrid macrophages that bolster antibacterial immunity and simultaneously limit inflammation, reducing disease burden and promoting host survival.

Preparation of Calcipotriol Emulsion Using Bacterial Exopolysaccharides as Emulsifier for Percutaneous Treatment of Psoriasis Vulgaris.

An exopolysaccharides/calcipotriol (EPS/CPT) emulsion was prepared using bacterial EPS as emulsifier, sunflower oil as an oil phase and CPT as the loaded drug, and the effect of this emulsion on psoriasis vulgaris treatment was evaluated. An EPS composed of mannose (70.56%) and glucose (29.44%) was obtained from the marine mangrove bacteria Bacillus amyloliquefaciens ZWJ (Zhu Wenjing) strain. The EPS has significant emulsifying activity at the concentration of 1.5%. The prepared EPS/CPT emulsion has small and stable particle size, with a drug content of 0.00492%, and good spreading properties. The in vitro drug release results revealed that the emulsion showed a certain sustained release effect. In vitro and in vivo animal experiments show that the EPS/CPT emulsion can effectively treat psoriasis vulgaris by increasing the accumulation of CPT in psoriatic skin lesions and reducing the levels of inflammatory cells and inflammatory factors (TNF and IL6). Additionally, it has a certain effect on reducing the side effects associated with CPT. This study lays a foundation for the research of EPS in the topical application of medical materials and treatment of psoriasis.

Levofloxacin Hemihydrate In Situ Gelling Ophthalmic Solution: Formulation Optimization and In Vitro and In Vivo Evaluation.

Bacterial conjunctivitis is a leading cause of ocular infections requiring short-term therapeutic treatment with frequent administration of drugs on daily basis. Topical dosage forms available in the market for the treatment of bacterial conjunctivitis such as simple drug solutions and suspensions are rapidly eliminated from the precorneal space upon instillation due to tear turn over and nasolacrimal drainage, limiting intraocular bioavailability of drug to less than 10% of the administered dose. To overcome issues related to conventional drop, an effort was made to design and evaluate prolong release ophthalmic solution of levofloxacin hemihydrate (LFH) using ion-sensitive in situ gelling polymer. Gellan gum was used as the in situ gelling agent. Formulations were screened based on in vitro gelation time, in vitro drug release, and stability towards sol to gel conversion upon storage. The prototype formulations exhibiting quick in vitro gelling time (< 15 s), prolonged in vitro drug release (18-24 h), and stability for at least 6 months at 25°C/40% relative humidity (RH) and 40°C/25% RH were evaluated for pharmacokinetic studies using healthy New Zealand white rabbits. Tested formulations were found to be well-tolerated and showed significant increase in AUC0-24 (22,660.39 h ng/mL) and mean residence time (MRT 12 h) as compared with commercially available solution Levotop PF® (Ajanta Pharma Ltd., India)(AUC0-24 6414.63 h ng/mL and MRT 4 h). Thus, solution formulations containing in situ gelling polymer may serve as improved drug delivery system providing superior therapeutic efficacy and better patient compliance for the treatment of bacterial conjunctivitis.

Enhancement of levodopa stability when complexed with ß-cyclodextrin in transdermal patches.

Levodopa is a promising candidate for administration via the transdermal route because it exhibits a short plasma half-life and has a small window of absorption in the upper section of the small intestine. The aim of this study was to prepare stable levodopa transdermal patches. Both xanthan gum and Carbopol 971 polymers were selected with ethylcellulose constituting the backing layer of the prepared patches. The effect of adding ß-cyclodextrin on the prepared patches was investigated. The uniformity in thickness, weight and content of the studied patches was acceptable. Physicochemical characterization revealed that there was no interaction between levodopa and the applied polymer. The results proved that levodopa precipitated as an amorphous form in carbopol patches. Controlled drug release was achieved for all the tested patches over a 6 h period. However, increased permeation was achieved for the carbopol patches. Although cyclodextrin did not enhance levodopa permeation, the stability study confirmed that levodopa stability was enhanced when complexed with ß-cyclodextrin. The cumulative amount of drug released from carbopol patches is slightly higher than that of xanthan patches. The optimal stability was achieved in the carbopol/levodopa:ß-cyclodextrin patch. The levodopa-ß-cyclodextrin complex was successfully characterized using X-ray diffraction, NMR analysis and molecular dynamics simulations. In conclusion, carbopol/levodopa:ß-cyclodextrin patches can be considered as a promising stable and effective transdermal drug-delivery system.

The Use of Salmonella Typhim Vaccine to Diagnose Antibody Deficiency.

PURPOSE: The specific antibody response to the unconjugated 23-valent pneumococcal polysaccharide vaccine is one of the most common tests used to assess for possible humoral immunodeficiency. The results can be difficult to interpret because most people have been immunized with one or more of the pneumococcal vaccines and there is controversy regarding what constitutes a normal response. To circumvent this problem, we developed an ELISA to measure IgG-specific antibodies to the Salmonella Vi Typhim (S. Typhim) vaccine, a pure polysaccharide vaccine, which is a neoantigen for the vast majority of people in the USA. METHODS: We compared the pre- and post-vaccination serum titers to the Vi Typhim vaccine in healthy controls (n = 22), patients previously diagnosed with a primary immunodeficiency (n = 30), and patients referred for possible humoral immune deficiency (n = 29). We also determined if the S. Typhim vaccine could be used to assess specific antibody responses in people on antibody replacement therapy. RESULTS: Following immunization with the S. Typhim vaccine, we found that a 2-fold increase in titers is 100% sensitive and specific in detecting known humoral immune deficiencies as determined by ROC curve analysis. This cut-off value was successfully applied to possible immune deficiency patients (n = 29), resulting in the diagnosis of seven subjects with humoral immunodeficiency. The use of immunoglobulin replacement therapy did not affect the median response ratios compared to subjects not receiving gammaglobulin. CONCLUSION: This study suggests that measurement of the specific antibody response to the S. Typhim vaccine may have advantages over pneumococcal vaccination in the evaluation of the humoral immune response.

PD-1 suppresses protective immunity to Streptococcus pneumoniae through a B cell-intrinsic mechanism.

Despite the emergence of the programmed cell death 1 (PD-1):PD-1 ligand (PD-L) regulatory axis as a promising target for treating multiple human diseases, remarkably little is known about how this pathway regulates responses to extracellular bacterial infections. We found that PD-1(-/-) mice, as well as wild-type mice treated with a PD-1 blocking Ab, exhibited significantly increased survival against lethal Streptococcus pneumoniae infection following either priming with low-dose pneumococcal respiratory infection or S. pneumoniae-capsular polysaccharide immunization. Enhanced survival in mice with disrupted PD-1:PD-L interactions was explained by significantly increased proliferation, isotype switching, and IgG production by pneumococcal capsule-specific B cells. Both PD-L, B7-H1 and B7-DC, contributed to PD-1-mediated suppression of protective capsule-specific IgG. Importantly, PD-1 was induced on capsule-specific B cells and suppressed IgG production and protection against pneumococcal infection in a B cell-intrinsic manner. To our knowledge, these results provide the first demonstration of a physiologic role for B cell-intrinsic PD-1 expression in vivo. In summary, our study reveals that B cell-expressed PD-1 plays a central role in regulating protection against S. pneumoniae, and thereby represents a promising target for bolstering immunity to encapsulated bacteria.