Photo effect on the CD1d-binding ability of azobenzene-attached analogues of α-GalCer.

α-Galactosylceramide (α-GalCer) is recognized by the CD1d proteins on antigen-presenting cells at the ceramide moiety and the galactose moiety is presented to iNKT cells, which stimulates the immune responses. However, the immune suppression by repeated injections of α-GalCer has discouraged its development as an anti-cancer agent. To overcome the shortcoming by spatiotemporal restriction of its exposure, we synthesized the photochromic azobenzene-incorporated analogues and tested the photo-immunoregulation effect in its binding to CD1d. FACS analyses indicated that some of these analogues enhanced the affinity to CD1d on photo-irradiation by about 20%. A docking simulation suggests that the photochromic molecule should be bulkier for a clearer discrimination between on and off states.

Array comparative genomic hybridization analysis discloses chromosome copy number alterations as indicators of patient outcome in lymph node-negative breast cancer.

BACKGROUND: Patients with lymph node metastasis-negative (pN0) invasive breast cancer have favorable outcomes following initial treatment. However, false negatives which occur during routine histologic examination of lymph nodes are reported to underestimate the clinical stage of disease. To identify a high-risk group in pN0 invasive breast cancer, we examined copy number alterations (CNAs) of 800 cancer-related genes. METHODS: Using array-based comparative genomic hybridization (CGH) in 51 pN0 cases (19 relapsed and 32 non-relapsed cases), the positivities of specific gene CNAs in the relapsed and non-relapsed groups were compared. An unsupervised hierarchical cluster analysis was then performed to identify case groups that were correlated with patient outcomes. RESULTS: The cluster analysis identified three distinct clusters of cases: groups 1, 2, and 3. The major component was triple-negative cases (69%, 9 of 13) in group 1, luminal B-like (57%, 13 of 23) and HER2-overexpressing (26%, 6 of 23) subtypes in group 2, and luminal A-like subtype (60%, 9 of 15) in group 3. Among all 51 cases, those in group 1 showed significantly worse overall survival (OS) than group 2 (p = 0.014), and 5q15 loss was correlated with worse OS (p = 0.017). Among 19 relapsed cases, both OS and relapse-free survival (RFS) rates were significantly lower in group 1 than in group 2 (p = 0.0083 and 0.0018, respectively), and 5q15 loss, 12p13.31 gain, and absence of 16p13.3 gain were significantly correlated with worse OS and RFS (p = 0.019 and 0.0027, respectively). CONCLUSIONS: As the target genes in these loci, NR2F1 (5q15), TNFRSF1A (12p13.31), and ABCA3 (16p13.3) were examined. 5q15 loss, 12p13.31 gain, and absence of 16q13.3 gain were potential indicators of high-risk recurrence and aggressive clinical behavior of pN0 invasive breast cancers.

Evaluation of the impact of conventional immunosuppressant on the establishment of murine transplantation tolerance - an experimental study.

Regulatory T cells (Tregs) play a significant role in immune tolerance. Since Treg function deeply depends on Interleukin-2 signaling, calcineurin inhibitors could affect their suppressive potentials, whereas mammalian target of rapamycin (mTOR) inhibitors may have less impact, as mTOR signaling is not fundamental to Treg proliferation. We previously reported a novel mixed hematopoietic chimerism induction regimen that promotes Treg proliferation by stimulating invariant natural killer T cells under CD40 blockade. Here, we use a mouse model to show the impact of tacrolimus (TAC) or everolimus (EVL) on the establishment of chimerism and Treg proliferation in the regimen. In the immunosuppressive drug-dosing phase, peripheral blood chimerism was comparably enhanced by both TAC and EVL. After dosing was discontinued, TAC-treated mice showed gradual graft rejection, whereas EVL-treated mice sustained long-term robust chimerism. Tregs of TAC-treated mice showed lower expression of both Ki67 and cytotoxic T lymphocyte antigen-4 (CTLA-4), and lower suppressive activity in vitro than those of EVL-treated mice, indicating that TAC negatively impacted the regimen by interfering with Treg proliferation and activation. Our results suggest that the usage of calcineurin inhibitors should be avoided if utilizing the regimen to induce Tregs in vivo for the establishment of mixed hematopoietic chimerism.

Sublingual administration of liposomes enclosing alpha-galactosylceramide as an effective adjuvant of allergen immunotherapy in a murine model of allergic rhinitis.

BACKGROUND: Sublingual immunotherapy (SLIT) is an established efficacious approach for the treatment of allergic rhinitis (AR). However, SLIT requires a long administration period to establish stable and adequate responses. This study investigated the efficacy of the sublingual administration of an allergen with liposomes enclosing α-GalCer (α-GC-liposome) as a potential adjuvant in mice with AR. METHODS: Mice with AR induced by OVA received the sublingual administration of OVA, α-GC-liposomes, or OVA plus α-GC-liposomes for 7 days. After nasal re-challenge with OVA, nasal symptoms were evaluated. The serum levels of OVA-specific Ig, the cytokine production of CD4+ T cells in the cultures of cervical lymph node (CLN) cells, and the gene expression of CLNs were analyzed. RESULTS: Although IL-4, IL-5 and IL-13 production from CD4+ T cells in CLN cells was significantly inhibited by the sublingual administration of OVA alone in mice with AR induced by OVA, their nasal symptoms were not significantly diminished. However, the combined sublingual administration of α-GC-liposomes and OVA completely suppressed nasal symptoms, downregulated Th2 and Th17 type cytokine production in CD4+ T cells as well as Th2 and Th17 gene expressions, and upregulated Th1 type cytokine production as well as Th1 gene expressions in CLN cells. Additionally, the serum levels of specific IgG2a were promoted, and specific IgE and IgG1 were inhibited. CONCLUSIONS: Our findings suggest that the sublingual administration of an allergen with α-GC-liposomes as an adjuvant might increase the therapeutic efficacy and effectiveness of this treatment method.

iNKT cell activation plus T-cell transfer establishes complete chimerism in a murine sublethal bone marrow transplant model.

Transplant tolerance induction makes it possible to preserve functional grafts for a lifetime without immunosuppressants. One powerful method is to generate mixed hematopoietic chimeras in recipients by adoptive transfer of donor-derived bone marrow cells (BMCs). In our murine transplantation model, we established a novel method for mixed chimera generation using sublethal irradiation, CD40-CD40L blockade, and invariant natural killer T-cell activation. However, numerous BMCs that are required to achieve stable chimerism makes it difficult to apply this model for human transplantation. Here, we show that donor-derived splenic T cells could contribute to not only the reduction of BMC usage but also the establishment of complete chimerism in model mice. By cotransfer of T cells together even with one-fourth of the BMCs used in our original method, the recipient mice yielded complete chimerism and could acquire donor-specific skin-allograft tolerance. The complete chimeric mice did not show any remarks of graft versus host reaction in vivo and in vitro. Inhibition of the apoptotic signal resulted in increase in host-derived CD8+ T cells and chimerism brake. These results suggest that donor-derived splenic T cells having veto activity play a role in the depletion of host-derived CD8+ T cells and the facilitation of complete chimerism.

Donor bone marrow cells are essential for iNKT cell-mediated Foxp3+ Treg cell expansion in a murine model of transplantation tolerance.

Mixed chimerism induction is the most reliable method for establishing transplantation tolerance. We previously described a novel treatment using a suboptimal dose of anti-CD40 ligand (anti-CD40L) and liposomal formulation of a ligand for invariant natural killer T cells administered to sub-lethally irradiated recipient mice after donor bone marrow cell (BMC) transfer. Recipient mice treated with this regimen showed expansion of a Foxp3-positive regulatory T(Treg) cell phenotype, and formation of mixed chimera. However, the mechanism of expansion and bioactivity of Treg cells remains unclear. Here, we examine the role of donor BMCs in the expansion of bioactive Treg cells. The mouse model was transplanted with a heart allograft the day after treatment. The results showed that transfer of spleen cells in place of BMCs failed to deplete host interferon (IFN)-γ-producing CD8+ T cells, expand host Ki67+ CD4+ CD25+ Foxp3+ Treg cells, and prolong graft survival. Severe combined immunodeficiency mice who received Treg cells obtained from BMC-recipients accepted skin grafts in an allo-specific manner. Myeloid-derived suppressor cells, which were a copious cell subset in BMCs, enhanced the Ki67 expression of Treg cells. This suggests that donor BMCs are indispensable for the expansion of host bioactive Treg cells in our novel treatment for transplant tolerance induction.

Characterization and evaluation of stabilized particulate formulations as therapeutic oral vaccines for allergy.

Allergic conditions affect more than a quarter of the population in developed countries, but currently available treatments focus more on symptom relief than treating the underlying atopic condition. α-Galactosylceramide (α-GalCer) is a potent immunomodulating compound that has been shown to have a regulatory effect when delivered systemically in nanoparticles. Parenteral delivery is not preferred for chronic conditions, such as allergy, and therefore, the aim of this study was to determine whether a regulatory response could be induced through oral administration in a model of atopy through incorporation of α-GalCer into stable particulate formulations (cationic liposomes, polymerized liposomes and poly(lactic-co-glycolic acid) nanoparticles (PLGA NPs)). The formulations showed only minor changes in particle size, polydispersity index and retention of the model antigen ovalbumin (OVA) during incubation in simulated gastrointestinal (GI) conditions. Oral delivery of α-GalCer in cationic liposomes could induce immunostimulating effects systemically, as seen through increases in serum IgG antibody levels, whereas delivery of α-GalCer in polymerized liposomes and PLGA NPs induced local cytokine changes in the mesenteric lymph nodes (MLNs). The generated responses did not exhibit tolerogenic traits which could be useful for immunoregulation, but the responses generated varied between formulations and suggests that further characterization and optimization could lead to the desired immune response.

Increased Foxp3+Helios+ Regulatory T Cells and Decreased Acute Graft-versus-Host Disease after Allogeneic Bone Marrow Transplantation in Patients Receiving Sirolimus and RGI-2001, an Activator of Invariant Natural Killer T Cells.

Regulatory T (Treg) cells play a central role in immune tolerance and prevention of aberrant immune responses. Several studies have suggested that the risk of graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT) can be ameliorated by increasing Tregs. We have developed an approach of in vivo expansion of Tregs with RGI-2001, a novel liposomal formulation of a synthetic derivative of alpha-galactosylceramide, a naturally occurring ligand that binds to CD1 and activates and expands invariant natural killer cells. In preclinical studies, a single intravenous infusion of RGI-2001 expanded Treg and could ameliorate GVHD in a mouse model of allogeneic HCT. To explore the role of RGI-2001 in clinical HCT, we initiated a phase 2A clinical trial (n = 29), testing 2 different doses of RGI-2001 administered as a single infusion on day 0 of allogeneic HCT. RGI-2001 was well tolerated and without infusion reactions or cytokine release syndrome. A subset of patients (8 of 29, 28%) responded to RGI-2001 by inducing a markedly increased number of cells with a Treg phenotype. The Treg had a high Ki-67 index and were almost exclusively Helios+ and Foxp3+, indicating that their accumulation was due to expansion of natural Treg. Notably, the incidence of grade 2 to 4 GVHD in the 8 patients who responded to RGI-2001 was 12.5%, compared with 52.4% in the 21 patients who did not respond. No grade 3 or 4 GVHD was observed in the responder group, compared with a 9.5% incidence among nonresponders. Immunosuppression with sirolimus was also associated with a low incidence of GVHD, suggesting that RGI-2001 may have synergized with sirolimus to promote Treg expansion.

KLRG+ invariant natural killer T cells are long-lived effectors.

Immunological memory has been regarded as a unique feature of the adaptive immune response mediated in an antigen-specific manner by T and B lymphocytes. However, natural killer (NK) cells and γδT cells, which traditionally are classified as innate immune cells, have been shown in recent studies to have hallmark features of memory cells. Invariant NKT cell (iNKT cell)-mediated antitumor effects indicate that iNKT cells are activated in vivo by vaccination with iNKT cell ligand-loaded CD1d(+) cells, but not by vaccination with unbound NKT cell ligand. In such models, it previously was thought that the numbers of IFN-γ-producing cells in the spleen returned to the basal level around 1 wk after the vaccination. In the current study, we demonstrate the surprising presence of effector memory-like iNKT cells in the lung. We found long-term antitumor activity in the lungs of mice was enhanced after vaccination with iNKT cell ligand-loaded dendritic cells. Further analyses showed that the KLRG1(+) (Killer cell lectin-like receptor subfamily G, member 1-positive) iNKT cells coexpressing CD49d and granzyme A persisted for several months and displayed a potent secondary response to cognate antigen. Finally, analyses of CDR3ß by RNA deep sequencing demonstrated that some particular KLRG1(+) iNKT-cell clones accumulated, suggesting the selection of certain T-cell receptor repertoires by an antigen. The current findings identifying effector memory-like KLRG1(+) iNKT cells in the lung could result in a paradigm shift regarding the basis of newly developed extrathymic iNKT cells and could contribute to the future development of antitumor immunotherapy by uniquely energizing iNKT cells.

Co-localization of iron binding on silica with p62/sequestosome1 (SQSTM1) in lung granulomas of mice with acute silicosis.

The cellular mechanisms involved in the development of silicosis have not been fully elucidated. This study aimed to examine influence of silica-induced lung injury on autophagy. Suspensions of crystalline silica particles were administered transnasally to C57BL/6j mice. Immunohistochemical examination for Fas and p62 protein expression was performed using lung tissue specimens. Two-dimensional and quantitative analysis of silica deposits in the lungs were performed in situ using lung tissue sections by an in-air microparticle induced X-ray emission (in-air micro-PIXE) analysis system, which was based on irrradiation of specimens with a proton ion microbeam. Quantitative analysis showed a significant increase of iron levels on silica particles (assessed as the ratio of Fe relative to Si) on day 56 compared with day 7 (p<0.05). Fas and p62 were expressed by histiocytes in granulomas on day 7, and the expressions persisted for day 56. Fas- and p62-expressing histiocytes were co-localized in granulomas with silica particles that showed an increase of iron levels on silica particles in mouse lungs. Iron complexed with silica induces apoptosis, and may lead to dysregulations of autophagy in histiocytes of granulomas, and these mechanisms may contribute to granuloma development and progression in silicosis.

Blood-brain barrier pharmacoproteomics-based reconstruction of the in vivo brain distribution of P-glycoprotein substrates in cynomolgus monkeys.

The aim of this study was to investigate whether in vivo drug distribution in brain in monkeys can be reconstructed by integrating four factors: protein expression levels of P-glycoprotein (P-gp)/multidrug resistance protein 1 at the blood-brain barrier (BBB), in vitro transport activity per P-gp molecule, and unbound drug fractions in plasma and brain. For five P-gp substrates (indinavir, quinidine, loperamide, paclitaxel, and verapamil) and one nonsubstrate (diazepam), in vitro P-gp transport activities were determined by measuring transcellular transport across monolayers of cynomolgus monkey P-gp-transfected LLC-PK1 and parental cells. In vivo P-gp functions at the BBB were reconstructed from in vitro P-gp transport activities and P-gp expression levels in transfected cells and cynomolgus brain microvessels. Brain-to-plasma concentration ratios (Kp,brain) were reconstructed by integrating the reconstructed in vivo P-gp functions with drug unbound fractions in plasma and brain. For all compounds, the reconstructed Kp,brain values were within a 3-fold range of observed values, as determined by constant intravenous infusion in adult cynomolgus monkeys. Among four factors, plasma unbound fraction was the most sensitive factor to species differences in Kp,brain between monkeys and mice. Unbound brain-to-plasma concentration ratios (Kp,uu,brain) were reconstructed as the reciprocal of the reconstructed in vivo P-gp functions, and the reconstructed Kp,uu,brain values were within a 3-fold range of in vivo values, which were estimated from observed Kp,brain and unbound fractions. This study experimentally demonstrates that brain distributions of P-gp substrates and nonsubstrate can be reconstructed on the basis of pharmacoproteomic concept in monkeys, which serve as a robust model of drug distribution in human brain.

Terpendole E and its derivative inhibit STLC- and GSK-1-resistant Eg5.

Terpendole E is first natural product found to inhibit mitotic kinesin Eg5, but its inhibitory mechanism remains to be revealed. Here, we report the effects of terpendole E and 11ketopaspaline (a new natural terpendole E analogue) on the Eg5-microtubule interaction and in several Eg5 mutants. 11-Ketopaspaline is a shunt product from terpendole E, and it shows potent inhibitory activity against the microtubule-stimulated ATPase activity of Eg5. Unlike other Eg5 inhibitors, such as S-trityl-L-cysteine (STLC) and GSK-1, both terpendole E and 11-ketopaspaline only partially inhibited Eg5-microtubule interaction. Furthermore, terpendole E and 11-ketopaspaline inhibited several Eg5 mutants that are resistant to STLC (Eg5(D130A), Eg5(L214A)) or GSK-1 (Eg5(I299F), Eg5(A356T)), but with the same extent of inhibition against wild-type Eg5. Because Eg5(D130A) and Eg5(L214A) show cross-resistance to most known Eg5 inhibitors, which bind the L5 loop, these results suggest that terpendole E and its analogues have a different binding site and/or inhibitory mechanism to those for L5 loop-binding type Eg5 inhibitors.

Effects of Italian ryegrass silage supplementation on feed intake and behavior of pregnant sows.

This study aimed to assess the behavior and stress status of pregnant sows following supplementation with Italian ryegrass silage (IRS) and the impact of feeding the IRS on feeding costs. Six sows with an initial body weight (BW) of 238.6 ± 5.9 kg were allotted to a 6 × 3 Latin square design with a 5-day acclimatization period followed by a 5-day data collection period. A commercial diet was replaced by IRS on a dry matter (DM) basis up to 0%, 9%, and 13% in the control treatment and the two test treatments, respectively. Apart from collecting data on daily feed intake and BW, urine was collected, and video footage was recorded for the last day of each treatment for analysis of urinary cortisol and behavior. There were no leftovers with all diets and nutrient uptake was unaffected (p > 0.05), while BW gain decreased (p < 0.05) to be a limited range from 1% to 3%, with increased inclusion of IRS. Both the behavior of sows and cortisol concentration were unaffected (p > 0.05). Furthermore, it was estimated that feeding 13% DM of IRS would reduce feed costs by 17%. IRS would be acceptable in replacing up to 13% of the commercial diet and cutting feeding costs.

IL-21-induced Bepsilon cell apoptosis mediated by natural killer T cells suppresses IgE responses.

Epidemiological studies have suggested that the recent increase in the incidence and severity of immunoglobulin (Ig)E-mediated allergic disorders is inversely correlated with Mycobacterium bovis bacillus Calmette Guerin (BCG) vaccination; however, the underlying mechanisms remain uncertain. Here, we demonstrate that natural killer T (NKT) cells in mice and humans play a crucial role in the BCG-induced suppression of IgE responses. BCG-activated murine Valpha14 NKT cells, but not conventional CD4 T cells, selectively express high levels of interleukin (IL)-21, which preferentially induces apoptosis in Bepsilon cells. Signaling from the IL-21 receptor increases the formation of a complex between Bcl-2 and the proapoptotic molecule Bcl-2-modifying factor, resulting in Bepsilon cell apoptosis. Similarly, BCG vaccination induces IL-21 expression by human peripheral blood mononuclear cells (PBMCs) in a partially NKT cell-dependent fashion. BCG-activated PBMCs significantly reduce IgE production by human B cells. These findings provide new insight into the therapeutic effect of BCG in allergic diseases.

Visualization of focused proton beam dose distribution by atomic force microscopy using blended polymer films based on polyacrylic acid.

A simple and sensitive sub-micrometer scale method for visualization of the dose distribution of a focused proton beam (FPB) was developed, taking advantage of the formation of a bulky crosslinked structure induced by FPB irradiation of a common polymer and cross-linker, polyacrylic acid-N, N'-methylene bisacrylamide, blend film surface. The irradiated part of the film swelled as a peak, and the height of swelling increased with increasing FPB fluence. The film was used as a proton beam-sensitive polymer film by analysis of the irradiated film surface using atomic force microscopy. The method was successfully applied to confirm the FPB pattern. Typical misaligned spot shape of FPB gave clear 3-dimensional structures, and the half-solenoidal nanostructures are visualized clearly by use of crescent shaped beam.

Uptake and translocation of cesium-133 in napiergrass (Pennisetum purpureum Schum.) under hydroponic conditions.

The present study reports the potential remediation of cesium (Cs) using napiergrass, which produces the largest biomass among the herbaceous plants in hydroponic culture containing stable Cs (Cs-133) at concentrations of 50, 150, 300, 1000, and 3,000 µM using cesium chloride (CsCl), with 0 µM Cs as a control concentration. Plant height was significantly decreased in higher Cs-treated conditions (300, 1000, and 3000 µM Cs) at 7 weeks after treatment (WAT), but tiller numbers tended to increase compared with the control plant. No significant difference was observed in the aboveground dry matter weight in all Cs treatments throughout the study period. Cs content in the roots, leaf blades, and leaf sheaths clearly increased with increasing Cs concentration in the solutions. Cs content in the aboveground parts (leaf blades and leaf sheaths) was consistently higher than in the roots at concentration of 3,000 µM. Total Cs contents in the aboveground parts were 6305 and 26,365 mg kg(-1) at 7WAT in 1000- and 3000-µM Cs treatments, respectively. Mean values of transfer factors (TFs) in the aboveground parts were 50 µM=0.78, 150 µM=1.02, 300 µM=0.86, 1,000 µM=0.68, and 3,000 µM=0.94, respectively at 7WAT. Due to its high Cs content and high TF in the aboveground parts, napiergrass may be a candidate plant with high potential for phytoremediation of Cs from Cs-137-contaminated soil.

Chimerism through the activation of invariant natural killer T cells prolongs graft survival after transplantation of induced pluripotent stem cell-derived allogeneic cardiomyocytes.

The loss of functional cells through immunological rejection after transplantation reduces the efficacy of regenerative therapies for cardiac failure that use allogeneic induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Recently, mixed-chimera mice with donor-specific immunotolerance have been established using the RGI-2001 (liposomal formulation of α-galactosyl ceramide) ligand, which activates invariant natural killer T (iNKT) cells. The present study aimed to investigate whether mixed chimerism, established using RGI-2001, prolongs graft survival in allogeneic iPSC-CM transplantation. Mixed-chimera mice were established via combinatorial treatment with RGI-2001 and anti-CD154 antibodies in an irradiated murine bone marrow transplant model. Luciferase-expressing allogeneic iPSC-CMs were transplanted into mixed-chimera and untreated mice, followed by in vivo imaging. RGI-2001 enhanced iNKT cell activation in mice, and mixed chimerism was successfully established. In vivo imaging revealed that while the allografts were completely obliterated within 2 weeks when transplanted to untreated mice, their survivals were not affected in the mixed-chimera mice. Furthermore, numerous CD3+ cells infiltrated allografts in untreated mice, but fewer CD3+ cells were present in mixed-chimera mice. We conclude that mixed-chimera mice established using RGI-2001 showed prolonged graft survival after allogeneic iPSC-CM transplantation. This donor-specific immunotolerance might increase the efficacy of regenerative therapies for heart failure with allogeneic iPSC-CMs.

Pharmacologic expansion of donor-derived, naturally occurring CD4(+)Foxp3(+) regulatory T cells reduces acute graft-versus-host disease lethality without abrogating the graft-versus-leukemia effect in murine models.

Adoptive transfer of regulatory T cells (Tregs) prevents graft-versus-host disease (GVHD) in mouse models, indicating a pivotal role for Tregs in controlling GVHD. The present study demonstrates the efficacy of Tregs pharmacologically induced in vivo in GVHD prevention. A single i.v. administration of a liposomal formulation of α-galactosylceramide (RGI-2001) at the time of allogeneic bone marrow transplantation with spleen cells significantly prolonged the survival of mice experiencing lethal acute GVHD. RGI-2001 expanded donor-derived CD4(+)Foxp3(+) Tregs in the spleen, lymph nodes, and bone marrow in a dose-dependent manner. On day 15 posttransplantation, the spleens of mice treated with RGI-2001 (1 µg/kg) contained 5-fold higher percentages or 10-fold higher numbers of CD4(+)Foxp3(+) Tregs compared with the spleens of untreated mice. Host-specific immunosuppression was introduced in treated mice, whereas the responsiveness to third-party alloantigens and leukemia cells was maintained. Using Foxp3:GFP reporter mice as donors, it was clearly shown that RGI-2001 expanded the pre-existing naturally occurring Tregs (nTregs) in donor spleen cells. Finally, RGI-2001 synergized with a subtherapeutic dose of rapamycin in nTreg expansion and further prolonged survival. Our results provide the first demonstration of the efficacy of nTregs pharmacologically expanded in vivo in preventing acute GVHD without abrogation of the beneficial graft-versus-leukemia effect.

Elemental analysis of lung tissue particles and intracellular iron content of alveolar macrophages in pulmonary alveolar proteinosis.

BACKGROUND: Pulmonary alveolar proteinosis (PAP) is a rare disease occurred by idiopathic (autoimmune) or secondary to particle inhalation. The in-air microparticle induced X-ray emission (in-air micro-PIXE) system performs elemental analysis of materials by irradiation with a proton microbeam, and allows visualization of the spatial distribution and quantitation of various elements with very low background noise. The aim of this study was to assess the secondary PAP due to inhalation of harmful particles by employing in-air micro-PIXE analysis for particles and intracellular iron in parafin-embedded lung tissue specimens obtained from a PAP patient comparing with normal lung tissue from a non-PAP patient. The iron inside alveolar macrophages was stained with Berlin blue, and its distribution was compared with that on micro-PIXE images. RESULTS: The elements composing particles and their locations in the PAP specimens could be identified by in-air micro-PIXE analysis, with magnesium (Mg), aluminum (Al), silicon (Si), phosphorus (P), sulfur (S), scandium (Sc), potassium (K), calcium (Ca), titanium (Ti), chromium (Cr), copper (Cu), manganase (Mn), iron (Fe), and zinc (Zn) being detected. Si was the major component of the particles. Serial sections stained by Berlin blue revealed accumulation of sideromacrophages that had phagocytosed the particles. The intracellular iron content of alveolar macrophage from the surfactant-rich area in PAP was higher than normal lung tissue in control lung by both in-air micro-PIXE analysis and Berlin blue staining. CONCLUSION: The present study demonstrated the efficacy of in-air micro-PIXE for analyzing the distribution and composition of lung particles. The intracellular iron content of single cells was determined by simultaneous two-dimensional and elemental analysis of paraffin-embedded lung tissue sections. The results suggest that secondary PAP is associated with exposure to inhaled particles and accumulation of iron in alveolar macrophages.

Production of both IL-27 and IFN-gamma after the treatment with a ligand for invariant NK T cells is responsible for the suppression of Th2 response and allergic inflammation in a mouse experimental asthma model.

Using an allergen-induced airway inflammation model, we show that an injection of alpha-galactosylceramide (alpha-GalCer), a ligand for invariant NK T (iNKT) cells, induced IL-27 and that this process is essential for the attenuation of the Th2 response. After the systemic administration of alpha-GalCer into the mice primed with OVA in alum, Th2 cytokine production of OVA-primed CD4(+) T cells in their lymph nodes, IgG1 and IgE Ab formation, and infiltration of eosinophils in bronchoalveolar lavage after the OVA challenge were suppressed. Systemic administration of rIFN-gamma into OVA-primed mice could not reproduce these effects of alpha-GalCer. IL-27p28 was detected both in the culture supernatant of alpha-GalCer-stimulated spleen cells and in the serum of the alpha-GalCer-treated mice, but not in the iNKT cell-deficient mice. Splenic iNKT cells produced IL-27p28 in the culture supernatant upon stimulation with PMA plus ionomycin, although the transcript of IL-27p28 in the iNKT cells was constitutively expressed regardless of the stimulation. By contrast, the transcript of IL-27EBI3 was induced in the iNKT cells upon stimulation with PMA plus ionomycin in vitro and with alpha-GalCer treatment in vivo, suggesting that IL-27 (p28/EBI3) could be produced by iNKT cells in an activation-dependent manner. Although repeated injections of rIL-27 did not substitute for the effects of a single injection of alpha-GalCer, administration of rIL-27 along with rIFN-gamma reproduced in vivo effects of the alpha-GalCer injection. These data indicate that production of both IL-27 and IFN-gamma by the alpha-GalCer treatment is responsible for suppression of the Th2 response and allergic inflammation.