Culture insert device with perfusable microchannels enhancesin vitroskin model development and barrier function assessment.

The ever-stricter regulations on animal experiments in the field of cosmetic testing have prompted a surge in skin-related research with a special focus on recapitulation of thein vivoskin structurein vitro. In vitrohuman skin models are seen as an important tool for skin research, which in recent years attracted a lot of attention and effort, with researchers moving from the simplest 2-layered models (dermis with epidermis) to models that incorporate other vital skin structures such as hypodermis, vascular structures, and skin appendages. In this study, we designed a microfluidic device with a reverse flange-shaped anchor that allows culturing of anin vitroskin model in a conventional 6-well plate and assessing its barrier function without transferring the skin model to another device or using additional contraptions. Perfusion of the skin model through vascular-like channels improved the morphogenesis of the epidermis compared with skin models cultured under static conditions. This also allowed us to assess the percutaneous penetration of the tested caffeine permeation and vascular absorption, which is one of the key metrics for systemic drug exposure evaluation.

A CTL-based liposomal vaccine capable of inducing protection against heterosubtypic influenza viruses in HLA-A*0201 transgenic mice.

The current vaccination strategy against influenza is to induce the production of antibodies directed against surface antigens of viruses. However, the frequent changes in the surface antigens of influenza viruses allow the viruses to avoid antibody-mediated immunity. On the other hand, it is known that cytotoxic T-lymphocyte (CTL) populations directed against internal antigens of influenza A virus are broadly cross-reactive to influenza virus subtypes. In the present study, liposomal conjugates with CTL epitope peptides derived from highly conserved internal antigens of influenza viruses were evaluated for their ability to protect against infection with influenza viruses. Liposomal conjugates with peptide M1 58-66, an HLA-A*0201-binding CTL epitope present within the amino-acid sequence of the M1 coding region, successfully induced antigen-specific CD8(+) T-cells and CTLs in HLA-A*0201-transgenic mice. Moreover, after nasal infection with either the H1N1 or H3N2 virus, viral replication in the lung was significantly inhibited in the immunized mice. These protective activities lasted at least 6months after the immunization. Thus, these results suggest that liposome-coupled CTL epitope peptides derived from highly conserved internal antigens of influenza viruses might be applicable to the development of vaccines that induce protection against infection with heterosubtypic influenza viruses.

Efficient induction of cytotoxic T lymphocytes specific for severe acute respiratory syndrome (SARS)-associated coronavirus by immunization with surface-linked liposomal peptides derived from a non-structural polyprotein 1a.

Spike and nucleocapsid are structural proteins of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) and major targets for cytotoxic T lymphocytes (CTLs). In contrast, non-structural proteins encoded by two-thirds of viral genome are poorly characterized for cell-mediated immunity. We previously demonstrated that nucleocapsid-derived peptides chemically coupled to the surface of liposomes effectively elicited SARS-CoV-specific CTLs in mice. Here, we attempted to identify HLA-A*0201-restricted CTL epitopes derived from a non-structural polyprotein 1a (pp1a) of SARS-CoV, and investigated whether liposomal peptides derived from pp1a were effective for CTL induction. Out of 30 peptides predicted on computational algorithms, nine peptides could significantly induce interferon gamma (IFN-gamma)-producing CD8(+) T cells in mice. These peptides were coupled to the surface of liposomes, and inoculated into mice. Six liposomal peptides effectively induced IFN-gamma-producing CD8(+) T cells and seven liposomal peptides including the six peptides primed CTLs showing in vivo killing activities. Further, CTLs induced by the seven liposomal peptides lysed an HLA-A*0201 positive cell line expressing naturally processed, pp1a-derived peptides. Of note, one of the liposomal peptides induced high numbers of long-lasting memory CTLs. These data suggest that surface-linked liposomal peptides derived from pp1a might offer an efficient CTL-based vaccine against SARS.

Highly efficient antiviral CD8+ T-cell induction by peptides coupled to the surfaces of liposomes.

In previous studies, we have demonstrated that liposomes with differential lipid components display differential adjuvant effects when antigens (Ags) are chemically coupled to their surfaces. When ovalbumin was coupled to liposomes made by using unsaturated fatty acids, it was found to be presented not only to CD4(+) T cells but also to CD8(+) T cells and induced cytotoxic T lymphocytes (CTLs) which effectively eradicated the tumor from mice. In this study, we coupled liposomes to immunodominant CTL epitope peptides derived from lymphocytic choriomeningitis virus (LCMV) and evaluated its potency as an antiviral vaccine. The intramuscular immunization of mice with the peptide-liposome conjugates along with CpG resulted in the efficient induction of antiviral CD8(+) T-cell responses which conferred complete protection against not only LCMV Armstrong but also a highly virulent mutant strain, clone 13, that establishes persistent infections in immunocompetent mice. The intranasal vaccination induced mucosal immunity effective enough to protect mice from the virus challenge via the same route. Complete protection was achieved in mice even when the Ag dose was reduced to as low as 280 ng of liposomal peptide. This form of vaccination with a single CTL epitope induced Ag-specific memory CD8(+) T cells in the absence of CD4(+) T-cell help, which could be shown by the complete protection of CD4-knockout mice in 10 weeks as well as by the analysis of recall responses. Thus, surface-linked liposomal peptide might have a potential advantage for the induction of antiviral immunity.

Synthetic peptides coupled to the surface of liposomes effectively induce SARS coronavirus-specific cytotoxic T lymphocytes and viral clearance in HLA-A*0201 transgenic mice.

We investigated whether the surface-linked liposomal peptide was applicable to a vaccine based on cytotoxic T lymphocytes (CTLs) against severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV). We first identified four HLA-A*0201-restricted CTL epitopes derived from SARS-CoV using HLA-A*0201 transgenic mice and recombinant adenovirus expressing predicted epitopes. These peptides were coupled to the surface of liposomes, and inoculated into mice. Two of the liposomal peptides were effective for peptide-specific CTL induction, and one of them was efficient for the clearance of vaccinia virus expressing epitopes of SARS-CoV, suggesting that the surface-linked liposomal peptide might offer an effective CTL-based vaccine against SARS.

Peptides coupled to the surface of a kind of liposome protect infection of influenza viruses.

In our previous study, OVA conjugated on the surface of a liposome, we termed Oleoyl liposome, which consisted of dioleoyl phosphatidyl choline, dioleoyl phosphatidyl ethanolamine, dioleoyl phosphatidyl glycerol acid and cholesterol in a 4:3:7:2 molar ratio, induced OVA-specific IgG antibody production but not OVA-specific IgE antibody production that is detrimental to the host. Furthermore, OVA(257-264)-Oleoyl liposome elicited CTL responses in the presence of CpG and rejected E.G7 tumors in mice. In this study we tested whether a peptide-Oleoyl liposome conjugates are capable of inducing protection against viral growth. Subcutaneous inoculation of NP(366-374)-Oleoyl liposome with CpG inhibited growth of influenza viruses in lungs of mice. Thus, surface-linked liposomal peptide might serve as an effective vaccine without detrimental effects in the presence of immune potentiators.

Antigen chemically coupled to the surface of liposomes are cross-presented to CD8+ T cells and induce potent antitumor immunity.

We have previously demonstrated that liposomes with differential lipid components display differential adjuvant effects when Ags are chemically coupled to their surfaces. In the present study, Ag presentation of liposome-coupled OVA was investigated in vitro, and it was found that OVA coupled to liposomes made using unsaturated fatty acid was presented to both CD4+ and CD8+ T cells, whereas OVA coupled to liposomes made using saturated fatty acid was presented only to CD4+ T cells. Confocal laser scanning microscopic analysis demonstrated that a portion of the OVA coupled to liposomes made using unsaturated, but not saturated fatty acid, received processing beyond the MHC class II compartment, suggesting that the degradation of OVA might occur in the cytosol, and that the peptides generated in this manner would be presented to CD8+ T cells via MHC class I. The ability to induce cross-presentation of an Ag coupled to liposomes consisting of unsaturated fatty acid was further confirmed by in vivo induction of CTL and by the induction of tumor eradication in mice; E.G7 tumors in mice that received combined inoculation with OVA(257-264)-liposome conjugates, CpG, and anti-IL-10 mAbs were completely eradicated. In those mice, the frequency of CD8+ T cells reactive with OVA(257-264) peptides in the context of H-2K(b) was significantly increased. These results suggested that, by choosing lipid components for liposomes, surface-coupled liposomal Ags might be applicable for the development of tumor vaccines to present tumor Ags to APCs and induce antitumor responses.

Induction of differential T-cell epitope by plain- and liposome-coupled antigen.

The T-cell receptors of CD4(+) T lymphocytes recognize immunogenic peptide sequences bound within the groove of MHC class II molecules, and the peptides that bind to these molecules are known to share common structural motifs. For example, OVA(323-339), an I-A(d)-binding peptide, involves a motif of the I-A(d) peptide-binding groove. In the present study, OVA peptides of up to 26-mer were sequentially synthesized and screened, and two additional I-A(d) binding OVA peptides, OVA(20-43) and OVA(264-286), were found to stimulate CD4(+) T cells of OVA-immune BALB/c mice. OVA(20-43) involved structural motifs of the I-A(d) peptide-binding groove, while OVA(264-286) did not. The ability of these three I-A(d) binding OVA peptides to induce antigen-specific cytokine production was compared among CD4(+) T cells of mice immunized either with alum-adsorbed OVA (OVA-alum) or OVA chemically coupled to the surface of liposome (OVA-liposome). CD4(+) T cells of mice immunized with OVA-alum produced more cytokines when stimulated with OVA(264-286) than with OVA(323-339), while CD4(+) T cells of mice immunized with OVA-liposome conjugates produced more cytokines when stimulated with OVA(323-339) than with OVA(264-286). OVA(20-43) induced production of comparable levels of cytokines in mice immunized either with OVA-alum or OVA-liposome. Confocal laser scanning microscopic analysis demonstrated that chemically coupled OVA and liposomes were colocalized in APCs until OVA received processing. Three-dimensional structural analysis demonstrated that both OVA(264-286) and OVA(323-339) were present on the surface of OVA, but OVA(20-43) was not. These results suggested that the chemical coupling of OVA to liposome affected antigen processing in APCs and thus resulted in the induction of differential T-cell epitopes as compared with those induced by plain OVA.

5-(4-Nitrophenyl)-2,4-pentadien-1-al (NPPD) used as a tracer by nonscientists at a simulated crime scene.

5-(4-Nitrophenyl)-2,4-pentadien-1-al (NPPD) was used as a tracer by nonscientists at a simulated crime scene. A card with both a plastic-coated smooth surface and a porous cellulose matrix paper surface was coated with a methanol solution containing 0.5mg/mL of NPPD. The card was touched with bare fingers and fingers covered by a cotton glove. A color-change protocol was then used to detect the presence of NPPD. The bare fingers or the fingers of gloves were swabbed with a cotton swab, or the parts of the glove that had touched the card were cut out. The swabs or the cloth pieces were dipped in methanol, a 0.1% methanol solution of naphthoresorcinol was added, and then concentrated hydrochloric acid was added. The observation of a red color at this point indicated a positive test. NPPD was easily observed in the experiments involving bare fingers, but no color change was observed from the swabbing of the cotton glove. However, when the cloth pieces cut from the fingers of the glove were subjected to the test, the red color was observed. In an attempt to enhance the sensitivity of the test, the volumes of the reagent solutions were reduced, but no improvement in sensitivity was obtained.

An increased adjuvanticity of liposomes by the inclusion of phosphatidylserine in immunization with surface-coupled liposomal antigen.

BACKGROUND: Exposure of phosphatidylserine (PS) on apoptotic cells is known to result in the enhanced recognition of apoptotic cells by phagocytes. By the inclusion of PS in the lipid component of liposomes, increased liposome immune adjuvant activity was expected. METHODS: In the present study, two different liposome preparations containing either PS, i.e. PS-liposome, or phosphatidylcholine (PC), i.e. PC-liposome, were made, and macrophage recognition, processing, and antigen presentation of surface-coupled liposomal antigen were compared. RESULTS: When ovalbumin-liposome conjugates were added to a culture of macrophages, enhanced recognition and processing of ovalbumin by the macrophages were observed by the inclusion of PS in the liposomes. The results correlated well with those regarding macrophage antigen presentation of liposome-coupled ovalbumin. Furthermore, in vivo immunization in mice with ovalbumin-liposome conjugates made with PS-liposomes induced a significantly higher level of anti-ovalbumin IgG antibody production than was induced by ovalbumin-liposome conjugates made with PC-liposomes. IgE-selective unresponsiveness was induced by ovalbumin-liposome conjugates regardless of the lipid components of liposomes. CONCLUSIONS: These results suggest that the inclusion of PS in liposomes enhances recognition and processing of surface-coupled liposomal antigen by macrophages, and increases liposome immune adjuvant activity.

Liposomes with differential lipid components exert differential adjuvanticity in antigen-liposome conjugates via differential recognition by macrophages.

We previously reported that liposomes having differential lipid components displayed differential adjuvant effects when antigen was coupled with liposomes via glutaraldehyde. In the present study, antigen-liposome conjugates prepared using liposomes having differential lipid components were added to the macrophage culture, and phagocytosis and the antigen digest of liposome-coupled antigen by macrophages were then investigated. Antigen presentation by macrophages to an antigen-specific T-cell clone was further investigated using the same conjugates. Antigen-liposome conjugates which induced higher levels of antibody production in vivo were recognized more often, and the liposome-coupled antigen was digested to a greater degree by macrophages than antigen-liposome conjugates which induced lower levels of antibody production. These results correlated closely with those regarding antigen presentation by macrophages; when antigen was coupled to liposomes showing higher adjuvant effect, macrophages cocultured with antigen-liposome conjugates activated antigen-specific T-cells at a higher degree. The concentration of OVA in the macrophage culture added as antigen-liposome conjugates was approximately 32 microg/mL. However, the extent of T-cell activation was almost equal to that when 800 microg/mL of soluble OVA was added to the culture. The results of the present study demonstrated that the adjuvant activity of liposomes observed primary in vivo correlated closely with the recognition of antigen-liposome conjugates and antigen presentation of liposome-coupled antigen by macrophages, suggesting that the adjuvant effects of liposomes are exerted at the beginning of the immune response, i.e., recognition of antigen by antigen-presenting cells.

Selective inhibition of systemic anti-OVA IgE production in response to oral pre-treatment with OVA-liposome conjugates.

BACKGROUND: We have previously reported that intraperitoneal injection with OVA-liposome conjugates induces OVA-specific and IgE-selective unresponsiveness in mice. METHODS: In the present study, the effects of oral pre-treatment with OVA-liposome conjugates or with plain OVA solution on anti-OVA IgG antibody production were investigated in mice after subsequent immunization with alum-adsorbed OVA. Control mice received only the immunization. RESULTS: The levels of serum anti-OVA IgG antibody in mice receiving oral administration of OVA-liposome were comparable to those in the control mice. However, in mice receiving oral administration of the same dose of plain OVA, levels of serum anti-OVA IgG antibody were significantly lower than those in control mice. Surprisingly, anti-OVA IgE antibody production was completely inhibited in mice receiving oral administration of OVA-liposome conjugates. Splenic CD4(+) T cells of mice receiving oral administration of OVA-liposome and those of control mice produced comparable levels of cytokines, while those of mice receiving oral administration of plain OVA solution produced significantly lower levels of cytokines than those in the other two groups. CONCLUSION: Orally administered OVA-liposome did not affect anti-OVA IgG production but did inhibit anti-OVA IgE antibody production, while orally administered OVA solution inhibited production of both IgG and IgE antibodies. These results suggest that antigen-liposome conjugates can possibly be orally administered in order to control antigen-specific IgE antibody production, without affecting IgG antibody production.

T cell-independent regulation of IgE antibody production induced by surface-linked liposomal antigen.

Control of IgE Ab production is important for the prevention of IgE-related diseases. However, in contrast to the existing information on the induction of IgE production, little is known about the regulation of the production of this isotype, with the exception of the well-documented mechanism involving T cell subsets and their cytokine products. In this study, we demonstrate an alternative approach to interfere with the production of IgE, independent of the activity of T cells, which was discovered during the course of an investigation intended to clarify the mechanism of IgE-selective unresponsiveness induced by surface-coupled liposomal Ags. Immunization of mice with OVA-liposome conjugates induced IgE-selective unresponsiveness without apparent Th1 polarization. Neither IL-12, IL-10, nor CD8(+) T cells participated in the regulation. Furthermore, CD4(+) T cells of mice immunized with OVA-liposome were capable of inducing Ag-specific IgE synthesis in athymic nude mice immunized with alum-adsorbed OVA. In contrast, immunization of the recipient mice with OVA-liposome did not induce anti-OVA IgE production, even when CD4(+) T cells of mice immunized with alum-adsorbed OVA were transferred. In the secondary immune response, OVA-liposome enhanced anti-OVA IgG Ab production, but it did not enhance ongoing IgE production, suggesting that the IgE-selective unresponsiveness induced by the liposomal Ag involved direct effects on IgE, but not IgG switching in vivo. These results suggest the existence of an alternative mechanism not involving T cells in the regulation of IgE synthesis.

Cholesterol inclusion in liposomes affects induction of antigen-specific IgG and IgE antibody production in mice by a surface-linked liposomal antigen.

In the previous study, we investigated the induction of ovalbumin (OVA)-specific antibody production in mice by OVA-liposome conjugates made using four different lipid components, including unsaturated carrier lipid and three different saturated carrier lipids. All of the OVA-liposome conjugates tested induced IgE-selective unresponsiveness. The highest titer of anti-OVA IgG was observed in mice immunized with OVA-liposomes made using liposomes with the highest membrane fluidity, suggesting that the membrane fluidity of liposomes affects their adjuvant effect. In this study, liposomes with five different cholesterol inclusions, ranging from 0% to 43% of the total lipid, were made, and the induction of OVA-specific antibody production by OVA-liposome conjugates was compared among these liposome preparations. In contrast to the results in the previous study, liposomes that contained no cholesterol and possessed the lowest membrane fluidity demonstrated the highest adjuvant effect for the induction of IgG antibody production. In addition, when the liposomes with four different lipid compositions were used, OVA-liposome conjugates made using liposomes that did not contain cholesterol induced significantly higher levels of anti-OVA IgG antibody production than did those made using liposomes that contained cholesterol and, further, induced significant production of anti-OVA IgE. These results suggest that cholesterol inclusion in liposomes affects both adjuvanticity for IgG production and regulatory effects on IgE synthesis by the surface-coupled antigen of liposomes.

Protection of monkeys against Shiga toxin induced by Shiga toxin-liposome conjugates.

BACKGROUND: We previously reported that the purified Shiga toxins (Stx) Stx1 and Stx2, when coupled with liposomes, induced substantial production of anti-Stx1 and anti-Stx2 IgG antibody, respectively, in mice. The levels of anti-Stx antibody in the sera of mice immune to Stx-liposome correlated well with the protection against subsequent challenge with Stx. Furthermore, mice immunized with a mixture of Stx1-liposome and Stx2-liposome were successfully protected against oral infection with cytotoxin-producing Escherichia coli O157:H7. METHODS: In this study, the induction of protection against Stx2 by Stx2-liposomes was evaluated in monkeys. RESULTS: Stx2-liposomes induced a substantial amount of anti-Stx2 IgG antibodies as well as Stx2 neutralizing antibodies in monkeys. Test monkeys were successfully protected against challenge with lethal doses of Stx2. Moreover, these monkeys showed no apparent symptoms, while nonimmunized control monkeys died within 4 days with hemorrhagic gastroenteritis and renal disorder. In addition, as shown by other cases involving antigen-liposome conjugates, Stx2-liposome did not induce anti-Stx2 IgE antibody production, though it stimulated the production of a substantial amount of anti-Stx2 IgG antibodies. CONCLUSION: These results suggest that Stx-liposome conjugates may serve as candidate vaccines to induce protection against death caused by cytotoxin-producing E. coli infection.