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The skin is an attractive alternative administration route for allergy vaccination, as the skin is rich in dendritic cells (DCs) and is easily accessible. In the skin multiple subsets of DCs with distinct roles reside at different depths. In this study antigen (=allergen for allergy) formulations were injected in ex vivo human skin in a depth-controlled manner by using a hollow microneedle injection system. Biopsies were harvested at the injection site, which were then cultured for 72 h. Subsequently, the crawled-out cells were collected from the medium and analyzed with flow cytometry. Intradermal administration of ovalbumin (OVA, model antigen) solution at various depths in the skin did not affect the migration and maturation of DCs. OVA was taken up efficiently by the DCs, and this was not affected by the injection depth. In contrast, Bet v 1, the major allergen in birch pollen allergy, was barely taken up by dermal DCs (dDCs). Antigens were more efficiently taken up by CD14+ dDCs than CD1a+ dDCs, which in turn were more efficient at taken up antigen than Langerhans cells. Subsequently, both OVA and Bet v 1 were formulated in cationic and anionic liposomes, which altered antigen uptake drastically following intradermal microinjection. While OVA uptake was reduced by formulation in liposomes, Bet v 1 uptake in dDCs was increased by encapsulation in both cationic and anionic liposomes. This highlights the potential use of liposomes as adjuvant in intradermal allergy vaccine delivery. In conclusion, we observed that antigen uptake after intradermal injection was not affected by injection depth, but varied between different antigens and formulation.

PURPOSE\nThe aim of this study was to investigate the depth-dependent intradermal immunogenicity of inactivated polio vaccine (IPV) delivered by depth-controlled microinjections via hollow microneedles (HMN) and to investigate antibody response enhancing effects of IPV immunization adjuvanted with CpG oligodeoxynucleotide 1826 (CpG) or cholera toxin (CT).\nMETHODS\nA novel applicator for HMN was designed to permit depth- and volume-controlled microinjections. The applicator was used to immunize rats intradermally with monovalent IPV serotype 1 (IPV1) at injection depths ranging from 50 to 550 μm, or at 400 μm for CpG and CT adjuvanted immunization, which were compared to intramuscular immunization.\nRESULTS\nThe applicator allowed accurate microinjections into rat skin at predetermined injection depths (50-900 μm), -volumes (1-100 μL) and -rates (up to 60 μL/min) with minimal volume loss (±1-2%). HMN-mediated intradermal immunization resulted in similar IgG and virus-neutralizing antibody titers as conventional intramuscular immunization. No differences in IgG titers were observed as function of injection depth, however IgG titers were significantly increased in the CpG and CT adjuvanted groups (7-fold).\nCONCLUSION\nIntradermal immunogenicity of IPV1 was not affected by injection depth. CpG and CT were potent adjuvants for both intradermal and intramuscular immunization, allowing effective vaccination upon a minimally-invasive single intradermal microinjection by HMN.

Dermal immunization using antigen-coated microneedle arrays is a promising vaccination strategy. However, reduction of microneedle sharpness and the available surface area for antigen coating is a limiting factor. To overcome these obstacles, a layer-by-layer coating approach can be applied onto pH-sensitive microneedles. Following this approach, pH-sensitive microneedle arrays (positively charged at coating pH 5.8 and nearly uncharged at pH 7.4) were alternatingly coated with negatively charged diphtheria toxoid (DT) and N-trimethyl chitosan (TMC), a cationic adjuvant. First, the optimal DT dose for intradermal immunization was determined in a dose-response study, which revealed that low-dose intradermal immunization was more efficient than subcutaneous immunization and that the EC50 dose of DT upon intradermal immunization is 3-fold lower, as compared to subcutaneous immunization. In a subsequent immunization study, microneedle arrays coated with an increasing number (2, 5, and 10) of DT/TMC bilayers resulted in step-wise increasing DT-specific immune responses. Dermal immunization with microneedle arrays coated with 10 bilayers of DT/TMC (corresponding with ± 0.6 μg DT delivered intradermally) resulted in similar DT-specific immune responses as subcutaneous immunization with 5 μg of DT adjuvanted with aluminum phosphate (8-fold dose reduction). Summarizing, the layer-by-layer coating approach onto pH-sensitive microneedles is a versatile method to precisely control the amount of coated and dermally-delivered antigen that is highly suitable for dermal immunization.

The purpose of this study was to investigate the effect of various repeated fractional intradermal dosing schedules of inactivated polio vaccine serotype 1 (IPV1) on IPV1-specific IgG responses in rats. By utilizing an applicator that allowed for precisely controlled intradermal microinjections by using a single hollow microneedle, rats were immunized intradermally with 5 D-antigen units (DU) of IPV1 at 150μm skin depth. This dose was administered as a bolus, or in a repeated fractional dosing schedule: 4 doses of 1.25 DU (1/4th of total dose) were administered on four consecutive days or every other day; 8 doses of 0.625 DU (1/8th of total dose) were administered on eight consecutive days; or 4 exponentially increasing doses (0.04, 0.16, 0.8 and 4 DU), either with or without an exponentially increasing CpG oligodeoxynucleotide 1826 (CpG) dose, were administered on four consecutive days. All of these fractional dosing schedules resulted in up to ca. 10-fold higher IPV1-specific IgG responses than intradermal and intramuscular bolus dosing. IPV1 combined with adjuvant CpG in exponential dosing did not significantly increase the IPV1-specific IgG responses further, which demonstrated that maximal responses were achieved by fractional dosing. In conclusion, repeated fractional intradermal IPV1 dosing leads to superior IPV1-specific IgG responses without the use of adjuvants. These results indicate that a controlled release delivery system for intradermal IPV1 delivery can potentiate IPV1-specific IgG responses.

The aim of this work was to coat pH-sensitive microneedle arrays with inactivated polio vaccine (IPV) particles and N-trimethyl chitosan chloride (TMC) via electrostatic interactions, and assess the immunogenicity of the vaccine after topical application of the coated microneedles in rats. The surface of 200 μm long microneedles was first chemically modified with pH-sensitive (pyridine) groups and then coated with negatively charged IPV and a positively charged polymer (TMC). To obtain a sufficient high antigen dose, 10 layers of IPV were alternately coated with TMC. The binding of IPV and TMC onto pH-sensitive microneedles was quantified and visualized by using fluorescently labeled TMC and IPV. The release of IPV and TMC from the microneedles was evaluated in ex vivo human skin by fluorescence and the immunogenicity of (unlabeled) IPV was assessed after topical application of the coated microneedles in rats. pH-sensitive microneedles were homogeneously coated with 10 layers of both IPV and TMC, resulting in 45 D antigen units IPV and 700 ng TMC per microneedle array. Fluorescence microscopy imaging revealed that both IPV and TMC were released into ex vivo human skin upon application of the coated microneedles. Finally, in vivo application of IPV-TMC-coated pH-sensitive microneedles in rats led to the induction of IPV specific antibody responses, illustrating that they are practically applicable. Topical administration of pH-sensitive microneedles coated with polyelectrolyte multinanolayers of antigens and oppositely charged polymers may be a useful approach for microneedle-based vaccination.

Aims: An outbreak of Shiga Toxin 2 (Stx-2) producing enterohemorrhagic E.coli (EHEC) O104:H4 infection in May 2011 caused enterocolitis and an unprecedented high 22% rate of hemolytic uremic syndrome (HUS). The monoclonal anti-C5 antibody Eculizumab (ECU) has been used experimentally in EHEC patients with HUS but treatment efficacy is uncertain. ECU can effectively prevent hemolysis in paroxysmal nocturnal hemoglobinuria (PNH) caused by a lack of complement-regulating CD55 and CD59 on blood cells. We hypothesized a low expression of CD55 and CD59, as seen in atypical HUS in PNH, might correlate with HUS development in EHEC patients. Methods: 76 EHEC patients (34: only gastrointestinal symptoms [GI], 23: HUS, 19: HUS and neurological symptoms[HUS/N]) and 12 healthy controls (HC) were tested for the expression of CD55 and CD59 on erythrocytes and leukocytes retrospectively. Additionally, the effect of Stx-2 on CD55 and CD59 expression on erythrocytes and leukocytes was studied ex vivo. Results: CD55 expression on erythrocytes was similar in all patient groups and HC while CD59 showed a significantly higher expression in HUS and HUS/N patients compared to HC and the GI group. CD55 and CD59 expression on leukocytes and their subsets were significantly higher in all patient groups compared to HC regardless of ECU-treatment. However, CD59 expression was significantly higher in HUS and HUS/N patients treated combined with plasma separation (PS) and ECU compared to HC. Adding Stx-2 ex vivo had no effect on CD55 and CD59 expression on leukocytes from HC or patients. Conclusion: HUS evolved independently from CD55 and CD59 expression on peripheral blood cells in EHEC O104:H4 infected patients. Our data point to a different mechanism within the complement system for HUS development in EHEC patients, when considering the original designated use of ECU to compensate protectively for the lack of CD55 and CD59 in PNH.

BackgroundAn outbreak of Shiga Toxin 2 (Stx-2) producing enterohemorrhagic and enteroaggregative E.coli (EAHEC) O104H4 infection in May 2011 caused enterocolitis and an unprecedented high 22% rate of hemolytic uremic syndrome (HUS). The monoclonal anti-C5 antibody Eculizumab (ECU) has been used experimentally in EAHEC patients with HUS but treatment efficacy is uncertain. ECU can effectively prevent hemolysis in paroxysmal nocturnal hemoglobinuria (PNH) caused by a lack of complement-regulating CD55 and CD59 on blood cells. We hypothesized a low expression of CD55 and CD59, as seen in PNH, might correlate with HUS development in EAHEC patients.Methods76 EAHEC patients (34 only gastrointestinal symptoms [GI], 23: HUS, 19: HUS and neurological symptoms [HUS/N]) and 12 healthy controls (HC) were tested for the expression of CD55 and CD59 on erythrocytes and leukocytes retrospectively. Additionally, the effect of Stx-2 on CD55 and CD59 expression on erythrocytes and leukocytes was studied ex vivo.ResultsCD55 expression on erythrocytes was similar in all patient groups and HC while CD59 showed a significantly higher expression in HUS and HUS/N patients compared to HC and the GI group. CD55 and CD59 expression on leukocytes and their subsets was significantly higher in all patient groups compared to HC regardless of treatment type. However, CD59 expression on erythrocytes was significantly higher in HUS and HUS/N patients treated combined with plasma separation (PS) and ECU compared to HC. Adding Stx-2 ex vivo had no effect on CD55 and CD59 expression on leukocytes from HC or patients.ConclusionHUS evolved independently from CD55 and CD59 expression on peripheral blood cells in EAHEC O104:H4 infected patients. Our data do not support a role for CD55 and CD59 in HUS development during EAHEC O104:H4 infection and point to a different mechanism within the complement system for HUS development in EAHEC patients.

Ionization reactions of anti-9-chloro-9-methoxybicyclo[4.2.l]nona-2,4,7-trie(n8e) proceed without skeletal rearrangements under conditions of short life. Rate constants were measured for the reaction of 8 and its more saturated analogues with pyridine. From the relatively low reaction rate of triene 8 it is concluded that the [4.2.1] cation is destabilized consequent to the homoantiaromatic interaction between the cationic center and the butadiene moiety. In addition to the kinetic results, the presence of this type of interaction is revealed by a NMR study of the 9-methoxybicyclo[4.2.1]nona-2,4-dien-9-cya1t ion (21) and the 1 I-methoxybicyclo[4.4. l]undeca-2,4,8-trien-ll-ylc ation (25) under conditions of long life, Le., superacid media. The IH and I3C NMR data point to an interaction of the cationic center with one of the double bonds of the butadiene moiety. Obviously the mode of homoconjugative interaction is controlled by orbital symmetry.

X-irradiation of a single crystal of thiophosphate O,O,O-triester of methyl ß-D-ribopyranoside at 77 K produces a s phosphoranyl radical anion (2) by electron capture among secondary radicals resulting from bond scission. The ESR parameters of (2) in correlation with the x-ray analysis of its precursor establish a TBP structure with the unpaired electron in apical position. The results suggest that in irradiated nucleic acid polymers, electrons can be trapped by phosphorus giving rise to a phosphorus-centered radical which subsequently induces bond break by a or ß scission generating thermodynamically more stable sugar and base radicals.

Das durch Auflosen von 7-Chlor-norbomadien (I) in flussigem SO2 entstehende Kation (II) geht mit Triphenylphosphin (III) keine 7-Substitution zu (IV) ein.

Background: Partner notification (PN) is an essential case-finding tool in the management of sexually transmitted infections (STIs). Yet, data on the effectiveness and factors impacting implementation of PN in the Netherlands are lacking. With the aim of further exploring and improving the PN process, the current study assessed perceived barriers and facilitators among health care professionals in the STI clinical setting. In particular, we explored the management of PN in young heterosexual patients diagnosed with Chlamydia trachomatis (Ct).Methods: We conducted semi-structured interviews among 22 health care professionals (response rate 52%) from 5 of the 8 national STI clinics in the Netherlands. We carried out qualitative content analysis using a framework approach. All participants were nurses, aged mid 20 s to late 50 s, and all but one were female.Results: All health care professionals felt comfortable discussing PN. Other perceived facilitators for PN included: time, one-on-one consultations, interviewing skills (i.e. Motivational Interviewing) and a proactive helping style. Important barriers were identified as: sub-optimal guidelines, inaccurate sexual history, a lack of feedback regarding the motivational strategies that were used, and the lack of feedback regarding overall PN effectiveness. The health care professionals placed an emphasis on the care and treatment of the individual index patient rather than on discussion of PN, or on motivating and helping patients to engage in PN.Conclusions: Health care professionals identified several barriers that need to be overcome, and facilitators which need to be maintained. Future efforts should concentrate on introducing PN protocols, providing feedback on both the effectiveness of strategies used by health care professionals, and on the PN process as a whole, and educating health care professionals about Motivational Interviewing strategies. Moreover, the possible implementation of an Internet-based PN system should be explored.

While the norbornen-7-ylium cation dissolved in liquid SO2 does not undergo 7-substitution with PPh3, the analogous reaction with the 7-methoxynorbornen-7-ylium cation produces the 7-methoxynorbornen-7-yl-triphenylphosphonium cation; the intermediate 7-triphenylphosphonio-norbornen-7-ylium dication could be detected.