Micro and Nano Material Carriers for Immunomodulation.

Modulation of the immune system through the use of micro and nano carriers offers opportunities in transplant tolerance, autoimmunity, infectious disease, and cancer. In particular, polymeric, lipid, and inorganic materials have been used as carriers of proteins, nucleic acids, and small drug molecules to direct the immune system toward either suppressive or stimulatory states. Current technologies have focused on the use of particulates or scaffolds, the modulation of materials properties, and the delivery of biologics or small drug molecules to achieve a desired response. Discussed are relevant immunology concepts, the types of biomaterial carriers used for immunomodulation highlighting their benefits and drawbacks, the material properties influencing immune responses, and recent examples in the field of transplant tolerance.

A cell-based microarray to investigate combinatorial effects of microparticle-encapsulated adjuvants on dendritic cell activation.

Experimental vaccine adjuvants are being designed to target specific toll-like receptors (TLRs) alone or in combination, expressed by antigen presenting cells, notably dendritic cells (DCs). There is a need for high-content screening (HCS) platforms to explore how DC activation is affected by adjuvant combinations. Presented is a cell-based microarray approach, "immunoarray", exposing DCs to a large number of adjuvant combinations. Microparticles encapsulating TLR ligands are printed onto arrays in a range of doses for each ligand, in all possible dose combinations. Dendritic cells are then co-localized with physisorbed microparticles on the immunoarray, adherent to isolated islands surrounded by a non-fouling background, and DC activation is quantified. Delivery of individual TLR ligands was capable of eliciting high levels of specific DC activation markers. For example, either TLR9 ligand, CpG, or TLR3 ligand, poly I:C, was capable of inducing among the highest 10% expression levels of CD86. In contrast, MHC-II expression in response to TLR4 agonist MPLA was among the highest, whereas either MPLA or poly I:C, was capable of producing among the highest levels of CCR7 expression, as well as inflammatory cytokine IL-12. However, in order to produce robust responses across all activation markers, adjuvant combinations were required, and combinations were more represented among the high responders. The immunoarray also enables investigation of interactions between adjuvants, and each TLR ligand suggested antagonism to other ligands, for various markers. Altogether, this work demonstrates feasibility of the immunoarray platform to screen microparticle-encapsulated adjuvant combinations for the development of improved and personalized vaccines.

Dietary forage concentration and particle size affect sorting, feeding behaviour, intake and growth of Chinese Holstein male calves.

The objective of study was to evaluate the effect of forage concentration (F:C) and forage particle length (FPL) on sorting, feeding behaviour, intake, growth and body measurements of growing calves. Twenty-eight weaned calves of body weight 156.79 ± 33.44 (mean ± SD) were used in 2 × 2 factorial arrangements with the factors FPL of hay grass (full and short) and hay grass concentrations (low, 50% and high, 65%). The treatments were as follows: full length (FL) with low F:C (50:50), FL with high F:C(65:35), short length (SL) with low F:C (50:50) and SL with high F:C (65:35). Increasing F:C and decreasing FPL enhanced sorting for short and fine particle and sorting against long particle (p < 0.05). Dry matter intake (DMI) was decreased by decreasing the FPL (p < 0.05). Increasing F:C (65:35) increased the DMI (p < 0.05). A positive interaction between FPL and F:C was found for (daily weight gain) DWG, weight gain (WG) and feed conversation ratio (FCR) (p < 0.05). In case of feeding behaviour, interaction for eating time and eating time per kilogram DM was present. Increasing the F:C increased the eating time in both FL and SL (p < 0.05). Chopping of hay had decreased the chewing time (p < 0.05). Increasing F:C increased chewing time per kilogram DMI. High F:C decreased the lying time (p < 0.05) in FL and SL treatments (p < 0.05). Increasing F:C reduced the overall abnormal behaviour (p < 0.05). These results suggested that animals performed better at higher F:C at SL diet. Intensity of sorting for short and fine particle and against long particle increased at higher F:C and SL diets. Eating time and eating time per kilogram DMI increased by increasing F:C level in both FL and SL treatments. Chewing time increased by increasing the FPL, while increasing the F:C enhanced the chewing time per kilogram DMI and reduced animal's abnormal behaviour.

New strategies of DLI in the management of relapse of hematological malignancies after allogeneic hematopoietic SCT.

DLI is an effective strategy for patients with recurrent hematological malignancies after allogeneic hematopoietic SCT (allo-HSCT). DLI has been widely applied to boost the graft vs tumor (GVT) or GVL effects. However, given the potentially severe complications associated with conventional DLI and transient GVL effect, new strategies for DLI are emerging. In this review, we have discussed the recent important studies on DLI as a prophylactic or therapeutic modality for relapsed hematological disorders after allo-HSCT. The strategies to separate GVL from GVHD have also been discussed. Leukemia-targeting therapy and lymphodepletion combined with DLI, and prophylactic DLI after allo-HSCT are often employed for patients with high risk of relapse, which has been reviewed as well. In addition, we have also discussed the issues on DLI to be further addressed, such as the doses, timing and frequency of DLI in different clinical settings, leukemic antigen-specific DLI as well as how to augment GVL effect while attenuating GVHD.

International Transporter Consortium commentary on clinically important transporter polymorphisms.

This Commentary focuses on genetic polymorphisms in membrane transporters. We present two polymorphisms for which there is a compelling body of literature supporting their clinical relevance: OATP1B1 (c.521T>C, p.V174A, rs4149056) and BCRP (c.421C>A, p.Q141K, rs2231142). The clinical evidence demonstrating their role in variation in pharmacokinetics and pharmacodynamics is described along with their allele frequencies in ethnic populations. Recommendations for incorporating studies of transporter polymorphisms in drug development are provided, along with the regulatory implications.

Drug transporters in drug efficacy and toxicity.

Drug transporters are now widely acknowledged as important determinants governing drug absorption, excretion, and, in many cases, extent of drug entry into target organs. There is also a greater appreciation that altered drug transporter function, whether due to genetic polymorphisms, drug-drug interactions, or environmental factors such as dietary constituents, can result in unexpected toxicity. Such effects are in part due to the interplay between various uptake and efflux transporters with overlapping functional capabilities that can manifest as marked interindividual variability in drug disposition in vivo. Here we review transporters of the solute carrier (SLC) and ATP-binding cassette (ABC) superfamilies considered to be of major importance in drug therapy and outline how understanding the expression, function, and genetic variation in such drug transporters will result in better strategies for optimal drug design and tissue targeting as well as reduce the risk for drug-drug interactions and adverse drug responses.

All-optical cascaded laser wakefield accelerator using ionization-induced injection.

We report on near-GeV electron beam generation from an all-optical cascaded laser wakefield accelerator (LWFA). Electron injection and acceleration are successfully separated and controlled in different LWFA stages by employing two gas cells filled with a He/O2 mixture and pure He gas, respectively. Electrons with a Maxwellian spectrum, generated from the first LWFA assisted by ionization-induced injection, were seeded into the second LWFA with a 3-mm-thick gas cell and accelerated to be a 0.8-GeV quasimonoenergetic electron beam, corresponding to an acceleration gradient of 187 GV/m. The demonstrated scheme paves the way towards the multi-GeV laser accelerators.

Generating quasi-single-cycle relativistic laser pulses by laser-foil interaction.

A scheme for producing nearly single-cycle relativistic laser pulses is proposed. When a laser pulse interacts with an overdense thin foil, because of self-consistent nonlinear modulation, the latter will be more transparent to the more intense part of the laser, so that a transmitted pulse can be much shorter than the incident pulse. Using two-dimensional particle-in-cell simulation and analytical modeling, it is found that a transmitted pulse of duration 4 fs and peak intensity 3 x 10{20} W/cm{2} can be generated from a circularly polarized laser pulse. The intensity of the resulting pulse is only limited by that of the incident pulse, since this scheme involves only laser-plasma interaction.

Dexamethasone induces IL-10-producing monocyte-derived dendritic cells with durable immaturity.

It is highly desirable that immature dendritic cells (DC) used for tolerance induction maintain steady immature state with predominant interleukin (IL)-10 production. In this study, we attempted to develop DC with durable immaturity and other tolerogenic features by using dexamethasone (Dex). We found DC derived from human monocytes in the presence of 10(-7) m Dex were negative for CD1a. Compared with control transduced DC (Ctrl-DC), Dex-DC expressed lower CD40, CD80 and CD86 but equivalent human leucocyte antigen-DR. Both immature Dex- and Ctrl-DC did not express CD83. Nevertheless, upon stimulation of lipopolysaccharide (LPS) or CD40 ligand, the expression of CD40, CD80, CD83 and CD86 was upregulated on Ctrl-DC but not on Dex-DC. The immaturity of Dex-DC was durable following Dex removal. Interestingly, Dex-DC maintained production of large amount of IL-10 and little IL-12 five days after Dex removed. Further study indicated that high-level IL-10 production by Dex-DC was associated with high-level phosphorylation of extracellular signal-regulated kinase (ERK) as blockade of this enzyme markedly attenuated IL-10 production. Furthermore, Dex-DC sustained the capability of high phosphorylation of ERK and IL-10 production 5 days after Dex removal. In addition, Dex-DC had significantly lower activity in stimulating T-cell proliferation. Neutralization of IL-10, to some extent, promoted DC maturation activated by LPS, as well as T-cell stimulatory activity of Dex-DC. The above findings suggest that IL-10-producing Dex-DC with durable immaturity are potentially useful for induction of immune tolerance.

Clinical study of Lipo PGE1-inhibiting platelet activation in acute rejection after kidney transplantation.

To observe the efficacy of the platelet activation inhibitor Lipo PGE1 therapy in the recovery of graft function after an acute rejection episode after kidney transplantation. Forty patients with acute rejection after kidney transplantation were randomly assigned into groups treated with or without Lipo PGE1. The expression levels of CD61, CD63, and PAC-1 on platelet surfaces were assayed by flow cytometry. The recovery time for graft function and 1-year patient and graft survival rates were recorded. Compared with controls, the expression levels of CD61, CD63, and PAC-1 were lower among acute rejection patients who received Lipo PGE1 therapy. The recovery time for graft function was shorter and the 1-year patient and graft survival rates higher. Lipo PGE1 therapy in patients with acute rejection episodes may inhibit platelet activation thereby benefiting graft functional recovery. The 1-year survival rates of patients and grafts might be increased if the expression levels of CD61, CD63, and PAC-1 on the platelet surfaces was decreased by Lipo PGE1 therapy.

Suppression of interleukin-12 production through endogenously secreted interleukin-10 in activated dendritic cells: involvement of activation of extracellular signal-regulated protein kinase.

Our recent study suggested the reverse relationship between the production of interleukin-10 (IL-10) and IL-12 in dendritic cells (DCs) activated by lipopolysaccharide (LPS) or LPS plus interferon (IFN)-gamma. In the present study, a series of experiments were performed to investigate the mechanisms responsible for this reverse relationship. Our results showed that neutralization of the secreted IL-10 by antibody could enhance the production of IL-12. Neutralization of IL-12 by antibody did not affect the IL-10 production. Addition of exogenous IL-10 suppressed the production of IL-12 by activated DCs, and addition of exogenous IL-12 did not affect IL-10 production. TaqMan real-time reverse transcriptase-polymerase chain reaction supported the fact that the observed effects occurred at mRNA transcription level. We also found that LPS or LPS plus IFN-gamma significantly enhanced the phosphorylation of extracellular signal-regulated protein kinase (ERK) and p38 mitogen-activated protein kinase. In addition, inhibition of ERK by PD98059 significantly suppressed IL-10 and increased the IL-12 production. Exogenous IL-10 reversed the upregulated production of IL-12 induced by PD98059. The above findings suggest a unidirectional negative autocrine regulation of IL-12 by IL-10 in activated DCs and that activation of ERK involves the differential production of IL-10 and IL-12 by activated DCs. Thus, the regulation of differential production of IL-10 and IL-12 may play an important role for DCs in priming T helper 1 (Th1) or Th2 in the immune responses.

Relationship between the expression levels of CD61, CD63, and PAC-1 on platelet surface in peripheral blood and the transplanted kidney function.

OBJECTIVE: To analyze the relationships between the expression levels of CD61, CD63, and PAC-1 on the platelet surface and the incidences of acute rejection and tubular necrosis as well as the recovery of graft function after renal transplantation. METHODS: The expression levels of CD61, CD63, and PAC-1 on platelet surfaces were assayed by flow cytometry in 86 patients with different stages of uremia before and after transplantation. Patients were divided into three groups: 29 patients with normal graft function, 30 with acute rejection, and 27 with acute tubular necrosis. Patients with acute rejection were randomly assigned into groups treated with or without anticoagulants. RESULTS: The expression levels of CD61, CD63, and PAC-1 on platelet surfaces significantly increased (P <.05) among patients with acute rejection, as compared with those with normal graft function or acute tubular necrosis. Compared with controls, the expression levels of CD61, CD63, and PAC-1 were lower among acute rejection patients who, received anticoagulant therapy. The recovery time for graft function shorter and, the 1-year patients and graft survival rates higher. CONCLUSIONS: The pretransplant expression levels of CD61, CD63, and PAC-1 on platelet surface were significantly higher among patients with acute rejection, suggesting that this complication rather than acute tubular necrosis may be related to platelet activation. Patients with acute rejection displayed benefit from anticoagulant therapy.

Monocyte-derived CD1a+ dendritic cells generated in two different culture systems: immunophenotypic and functional comparison.

Previous studies demonstrated that CD1a+ dendritic cells (DCs) could not be prepared ex vivo without using fetal calf serum (FCS). Recently, we developed a method of using heparin to induce differentiation of human monocytes into CD1a+ DCs without using FCS. In order to determine the potential clinical applicability of heparin-induced CD1a+ DCs, we conducted this study to compare both types of CD1a+ DCs, immunophenotypically and functionally. Our results showed that the expression of CD1a on heparin-DCs was lower than that on FCS-DCs. Both types of DCs expressed similar levels of CD11c, HLA-DR, CD40, CD83, CD80 and CD86 before and after lipopolysaccharide stimulation. Immature heparin-DCs and FCS-DCs had similar phagocytic activities. Heparin-DCs consistently secreted higher interleukin-10 (IL-10) and lesser IL-12 than FCS-DCs after activation. Mature heparin-DCs were slightly more active than mature FCS-DCs in stimulating the proliferation of allogeneic CD4+ T cells. Both types of mature CD1a+ DCs primed the naïve CD4+ T cells to produce large amount of interferon-gamma (IFN-gamma). However, naïve CD4+ T cells stimulated with FCS-DCs produced more IFN-gamma, while the naïve CD4+ T cells stimulated with heparin-DCs produced more IL-5. The results indicate that both types of CD1a+ DCs do not have identical function in the priming of CD4+ T cells and have minor difference in immunophenotypes.

Tyrphostin-8 enhances transferrin receptor-mediated transcytosis in Caco-2- cells and inreases hypoglycemic effect of orally administered insulin-transferrin conjugate in diabetic rats.

PURPOSE: To investigate the effect of tyrphostin 8 (T-8), a GTPase inhibitor, on transferrin receptor (TfR)-mediated transcytosis of insulin-transferrin (In-Tf) conjugate in cultured enterocyte-like Caco-2 cells and on gastrointestinal (GI) absorption of In-Tf in streptozotocin (STZ)-induced diabetic rats. METHODS: Caco-2 cells and diabetic rats were used as in vitro and in vivo models, respectively. TfR-mediated transcytosis was measured using 125I-In-Tf. The absorption of insulin in diabetic rats was demonstrated by the hypoglycemic effect. Rat blood glucose level was determined using a ONE TOUCH blood glucose monitoring system. RESULTS: T-8 increased apical-to-basolateral transport of In-Tf conjugate by enhancing TfR-mediated transcytosis in filter-grown Caco-2 cell monolayer, and this enhancement was higher and faster than the previously reported brefeldin A (BFA)-induced effect. The measurement of transepithelial electrical resistance (TEER) during the transport study showed that T-8 was less destructive on the cell tight junction than BFA. The GI absorption of In-Tf was evaluated by its hypoglycemic effect after oral administration in STZ-induced diabetic rats. The glucose-lowering effect of orally administered In-Tf in STZ-induced diabetic rats was improved by either T-8 or BFA. However, the effect of T-8 was more potent than that of BFA, especially at 7 h after administration. Either non-conjugated insulin or insulin-human serum albumin (In-HSA) conjugate by itself or in combination with T-8 did not show any hypoglycemic effect after oral administration, indicating that T-8-enhanced hypoglycemic activity of In-Tf was due to a selective enhancement of TfR-mediated transcytosis. CONCLUSIONS: Our data indicated that T-8 could be used to increase the GI absorption of insulin as a transferrin conjugate. T-8, as an enhancer of TfR-mediated transcytosis, is better than the previously reported BFA. T-8 produces a higher increase on the transport of In-Tf and a lower toxicity on epithelial cells. Our findings provide an alternative approach to promote the GI absorption of insulin, as well as other peptide or protein drugs.

Hypoglycemic effect of insulin-transferrin conjugate in streptozotocin-induced diabetic rats.

Transferrin (Tf) receptor-mediated transcytosis of insulin-transferrin conjugate (In-Tf) has been demonstrated in cultured human enterocyte-like Caco-2 cells. In the present report, oral delivery of insulin as a Tf conjugate in streptozotocin (STZ)-induced diabetic rats was investigated. Human insulin was conjugated at a 1:1 molar ratio to iron-loaded human Tf by a disulfide linkage. The stability of In-Tf and the free insulin released from In-Tf was studied in the presence of rat liver slices by using radioimmunoassay. The release of free insulin involved a disulfide reduction reaction that was inhibited by the pretreatment of the liver slice with a sulfhydryl-reactive reagent N-ethylmaleimide. A protease inhibitor cocktail also showed a partial inhibition of insulin degradation. The biological activity of the conjugate was tested in STZ-induced diabetic rats with s.c. administration, and the conjugate exhibited a slow but prolonged hypoglycemic effect compared with that of the native human insulin. In-Tf also displayed a slow but prolonged hypoglycemic effect after oral administration in fasted STZ-induced diabetic rats in a dose-dependent manner. Furthermore, In-Tf was detected in the serum of rats at 4 h after oral administration of the conjugate, indicating that In-Tf can overcome the barriers in the gastrointestinal tract and be absorbed as an intact conjugate. These results demonstrate that transepithelial transport via TfR-mediated transcytosis is a feasible approach for developing the oral delivery of insulin, as well as other peptide drugs.

QSAR analysis of polyamine transport inhibitors in L1210 cells.

PURPOSE: In this paper, the authors attempt to construct a mathematical model to correlate the biological activities of 63 polyamine transport inhibitors in L1210 cells with their physicochemical parameters. METHOD: The inhibitory constants (Ki) were obtained from the published work of Bergeron et al. Non-weighted least square method was used in deriving the regression equations with a BMDP program. An AM1 subroutine of the HyperChem program was used to optimize the geometry and calculate the molecular dipole moments and the distance between two terminal amino groups. A CQSAR program was used to calculate Clog P (oct./w.). RESULTS: A good correlation (r2 = 0.81) was obtained by using a five-parameter equation including the distance between two terminal amino groups (d), the number of cationic charge (Charge), molecular weight (MW), dipole moment (mu), and hydrogen bond forming ability (Hb). CONCLUSION: This model accounts for 81% of the variance in the data and can be used to estimate transport-inhibitory activity of many other polyamine analogues. It gives some quantitative information about the relationship between the polyamine analogues' function as transport inhibitors and their molecular structures.