MHC-DRB alleles with amino acids Val11, Phe13, and the shared epitopes promote collagen-induced arthritis and a rapid IgG1 response in Filipino cynomolgus macaques.

Macaques are useful animal models for studying the pathogenesis of rheumatoid arthritis (RA) and the development of anti-rheumatic drugs. The purpose of this study was to identify the major histocompatibility complex (MHC) polymorphisms associated with the pathology of collagen-induced arthritis (CIA) and anti-collagen IgG induction in a cynomolgus macaque model, as MHC polymorphisms affect the onset of CIA in other animal models. Nine female Filipino cynomolgus macaques were immunized with bovine type II collagen (b-CII) to induce CIA, which was diagnosed clinically by scoring the symptoms of joint swelling over 9 weeks. MHC polymorphisms and anti-b-CII antibody titers were compared between symptomatic and asymptomatic macaques. Four of 9 (44%) macaques were defined as the CIA-affected group. Anti-b-CII IgG in the affected group increased in titer approximately 3 weeks earlier compared with the asymptomatic group. The mean plasma IgG1 titer in the CIA-affected group was significantly higher (p < 0.05) than that of the asymptomatic group. Furthermore, the cynomolgus macaque MHC (Mafa)-DRB1*10:05 or Mafa-DRB1*10:07 alleles, which contain the well-documented RA-susceptibility five amino acid sequence known as the shared epitope (SE) in positions 70 to 74, with valine at position 11 (Val11, V11) and phenylalanine at position 13 (Phe13, F13), were detected in the affected group. In contrast, no MHC polymorphisms specific to the asymptomatic group were identified. In conclusion, the presence of V11 and F13 along with SE in the MHC-DRB1 alleles seems essential for the production of IgG1 and the rapid induction of severe CIA in female Filipino cynomolgus macaques.

Targeting cathepsin B by cycloastragenol enhances antitumor immunity of CD8 T cells via inhibiting MHC-I degradation.

BACKGROUND: The loss of tumor antigens and depletion of CD8 T cells caused by the PD-1/PD-L1 pathway are important factors for tumor immune escape. In recent years, there has been increasing research on traditional Chinese medicine in tumor treatment. Cycloastragenol (CAG), an effective active molecule in Astragalus membranaceus, has been found to have antiviral, anti-aging, anti-inflammatory, and other functions. However, its antitumor effect and mechanism are not clear. METHODS: The antitumor effect of CAG was investigated in MC38 and CT26 mouse transplanted tumor models. The antitumor effect of CAG was further analyzed via single-cell multiomics sequencing. Target responsive accessibility profiling technology was used to find the target protein of CAG. Subsequently, the antitumor mechanism of CAG was explored using confocal microscopy, coimmunoprecipitation and transfection of mutant plasmids. Finally, the combined antitumor effect of CAG and PD-1 antibodies in mice or organoids were investigated. RESULTS: We found that CAG effectively inhibited tumor growth in vivo. Our single-cell multiomics atlas demonstrated that CAG promoted the presentation of tumor cell-surface antigens and was characterized by the enhanced killing function of CD8+ T cells. Mechanistically, CAG bound to its target protein cathepsin B, which then inhibited the lysosomal degradation of major histocompatibility complex I (MHC-I) and promoted the aggregation of MHC-I to the cell membrane, boosting the presentation of the tumor antigen. Meanwhile, the combination of CAG with PD-1 antibody effectively enhanced the tumor killing ability of CD8+ T cells in xenograft mice and colorectal cancer organoids. CONCLUSION: Our data reported for the first time that cathepsin B downregulation confers antitumor immunity and explicates the antitumor mechanism of natural product CAG.

Echinacea purpurea Extract Enhances Natural Killer Cell Activity In Vivo by Upregulating MHC II and Th1-type CD4+ T Cell Responses.

There are a number of factors that cause immune system disruption, including infection caused by foreign antigens and decreased immunity due to excessive exercise, and public interest in improving immunity is growing. In this study, we investigate the immunomodulatory effects of Echinacea purpurea (E) extract in C57BL/6N mice that were exposed to a forced swimming exercise. There were six experimental groups as follows: wild-type, forced swimming exercise control, positive control (red ginseng, 300 mg/kg), and E (50, 100, and 200 mg/kg b.w.) groups. The mice were administered the E extract for 2 weeks. We detected chicoric acid, the active substance of E, through high-performance liquid chromatography and evaluated changes in the following laboratory values in response to forced swimming exercise using flow cytometry and ELISA: the major histocompatibility complex (MHC), CD4+ and CD8+ T cells, Th1 and Th2 cytokines, natural killer (NK) cell activity, and number of leukocytes. Oral E intake increased levels of MHC II, CD4+ T cells, Th1 cytokines, and NK cell activity. In addition, E treatment increased B cell proliferation, leukocyte counts, and immunoglobulin levels. Taken together, these results suggest that the chicoric acid of E can improve immune response by controlling NK cell activity, which may be a useful function for immunomodulation systems.

Effects of Amino Acid-Bound Zinc and Manganese Feed Additives on MHC Haplotype Chickens Challenged with Infectious Bronchitis Coronavirus.

Major histocompatibility complex (MHC) congenic chicken lines have been used as a model to study infectious bronchitis virus (IBV) immune responses in chickens. Zinc (Zn) and manganese (Mn) are trace minerals that act as enzyme cofactors in cellular reactions. In addition, Zn is an important modulator of immune responses, especially in the respiratory tract. Zinc and Zn + Mn amino acid complex supplements were tested to alleviate the effects of an IBV challenge using relatively resistant and susceptible MHC congenic chicken lines. Prior to the challenge with IBV, the amino acid-bound supplements induced better weight gain in the IBV-resistant chicken line (331/B2) compared to the birds fed with the sulfate-delivered supplements. No body weight differences were detected between IBV-challenged and unchallenged 331/B2 birds supplemented with Zn in amino acid complex. A reduction of respiratory signs was observed in 335/B19 birds fed with the diet supplemented with Zn in amino acid complexes at 4 dpi. Compared to the sulfate-bound trace minerals, 331/B2 chickens fed with the amino acid-bound supplements presented milder clinical sign trends at 6 dpi and less severe airsacculitis at 14 dpi. The total antibody response in serum in 331/B2 birds fed with the amino acid-bound Zn ration was the highest among all groups tested. Both amino acid-delivered trace mineral supplements induced a slightly higher antibody response than the sulfate-bound ration in both chicken lines. This experiment provides insights into the effect of Zn and Mn on the immunity of chickens with known different susceptibilities to IBV.

Immunomodulatory Effect of a Salvia plebeia R. Aqueous Extract in Forced Swimming Exercise-induced Mice.

This study investigated the immunomodulatory effect of Salvia plebeia R. aqueous extract (FIE-SP, SPW) in forced swimming exercise-induced mice and the immunostimulatory effects on Raw264.7 cells. Mice were randomly assigned to four groups: the control group (CON), the forced swimming test group (FST), and two FIE-SP groups (low and high dose of FIE-SP). Compared with the control group, the FIE-SP groups showed significantly increased ratios of T lymphocyte surface markers CD4+/CD8+ and major histocompatibility complex (MHC)I/MHCII, as well as increased concentrations of immunoglobulin (Ig)A and IgG. FIE-SP groups significantly increased Th1 cytokines and decreased Th2 cytokines compared with negative control exercise-induced mice. Conversely, the immunostimulatory effects of FIE-SP significantly increased phagocytic activities, nitric oxide (NO) production, and pro-inflammatory cytokines tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1ß in Raw264.7 cells. Furthermore, FIE-SP increased natural killer (NK) cell activities and cytokines (IL-12) in splenocytes compared with the CON group. These results indicated that FIE-SP supplementation could prevent imbalanced immune states and produce immunostimulatory effects to support innate immunity.

The protective effect of trilobatin against isoflurane-induced neurotoxicity in mouse hippocampal neuronal HT22 cells involves the Nrf2/ARE pathway.

Long-term exposure to isoflurane may induce long-term developmental neurotoxicity and cognitive impairments in the neonatal brains. Trilobatin, a leaf extract from the Chinese traditional sweet tea Lithocarpus polystachyus Rehd, possesses various biological properties including anti-inflammatory and anti-oxidant properties. Our study aimed to explore the neuroprotective effect of trilobatin on isoflurane-induced neurotoxicity in mouse hippocampal neuronal HT22 cells. The effects of trilobatin on cell viability, LDH release, apoptosis, and caspase-3/7 activity in isoflurane-induced HT22 cells were explored by CCK-8, LDH release assay, flow cytometry analysis, and caspase-3/7 activity assay, respectively. Oxidative stress was evaluated by measuring the levels of reactive oxygen species (ROS) and malonyldialdehyde (MDA) and activities of superoxide dismutase (SOD) and catalase (CAT). The expression of nuclear erythroid-2 related factor 2 (Nrf2), nuclear Nrf2, heme oxygenase-1 (HO-1), and NAD(P)H: quinone oxidoreductase 1 (NQO1) was determined by western blot and qRT-PCR. Results suggested that exposure to isoflurane significantly reduced cell viability and increased LDH release, apoptotic rate and caspase-3/7 activity in HT22 cells, which were abolished by trilobatin. Trilobatin reversed isoflurane-induced increase of ROS and MDA levels and reduction of SOD and CAT activities in HT22 cells. Additionally, trilobatin promoted the nuclear translocation of Nrf2 as well as the mRNA and protein expression of HO-1 and NQO1 in HT22 cells exposed to isoflurane. Nrf2 knockdown attenuated the effects of trilobatin on isoflurane-induced viability reduction, LDH release, apoptosis, and oxidative stress in HT22 cells. Overall, trilobatin protected HT22 cells against isoflurane-induced neurotoxicity via activating the Nrf2/antioxidant response element (ARE) pathway.

A novel biscoumarin compound ameliorates cerebral ischemia reperfusion-induced mitochondrial oxidative injury via Nrf2/Keap1/ARE signaling.

Some phytochemical-derived synthetic compounds have been shown to improve neurological disorders, especially in ischemic stroke. In this study, we identified a novel biscoumarin compound, known as COM 3, which had substantial antioxidant effects in neurons. Next, we found that COM 3 occupies a critical binding site between the Nrf2 and Keap1 dipolymer, impairing the inhibitory effects of Keap1 on Nrf2, both of which play central roles in increasing endogenous antioxidant activity. We verified that COM 3 could increase the survival of neurons experiencing oxygen and glucose deprivation (OGD) from 51.1% to 77.2% when exposure to 2.5 and 10 µg/mL of COM 3, respectively. In addition, the same concentrations of COM 3 could reduce brain infarct volumes by 33.8%to13.7%, respectively, while also reducing the neurobehavioral score from 3.3 to 1.4 on average in mice with a middle cerebral artery occlusion (MCAO). COM 3 reduced neuronal death from 36.5% to 13.9% and apoptosis from 35.1% to 18.2%. In addition, COM 3 could improve the neuronal mitochondrial energy metabolism after experiencing oxidative stress caused by OGD or MCAO. The present study suggests that COM 3 protects against OGD in neurons and MCAO in mice by interfering with the structure of Keap1 to activate the nuclear transcription of Nrf2, which balances endogenous redox activity and restores mitochondrial function. Hence, COM 3 might be a potential therapeutic agent for ischemic stroke in the clinic.

Genetic diversity of 10 indigenous chicken ecotypes from Southern Highlands of Tanzania based on Major Histocompatibility Complex-linked microsatellite LEI0258 marker typing.

Unraveling the genetic diversity of livestock species is central to understanding their value and importance for conservation and improvement in diverse production environments. In developing countries, information on genetic attributes of many livestock species is unfortunately scanty to support well-informed decision-making upon relevant management strategies. This study aimed at investigating allelic variability, genetic diversity, and genetic relationships of 10 indigenous chicken ecotypes from Southern Highlands of Tanzania using the Major Histocompatibility Complex-linked LEI0258 marker. A total of 400 DNA samples, 40 per ecotype, were genotyped by capillary electrophoresis. Thirty different alleles with sizes ranging from 197 to 569 bp were determined. The number of alleles ranged from 17 (Itunduma) to 21 (Mbeya), with an average of 19.20 alleles per ecotype. Allelic polymorphism was further evaluated through genotyping by Sanger sequencing. Thirty-three DNA samples with different fragment sizes were re-amplified and their alleles sequenced to depict polymorphism based on a combination of two repeat regions at 12 and 13 bp, respectively, and flanking regions with SNP and indels. The repeat region at 13 bp appeared 1 to 28 times, whereas the region at 12 bp appeared 3 to 19 times in all sequenced fragments. The numbers of indels and SNP determined were 7 and 9, respectively. From capillary electrophoresis, the Chunya and Msimbazi ecotypes exhibited the highest genetic diversity (0.937), whereas the lowest value (0.910) was observed from the Mbarali ecotype, with an average of 0.925. The Namtumbo and Wanging'ombe ecotypes showed high inbreeding coefficients (FIS > 0.05), whereas a high excess heterozygote value (FIS = -0.098) was observed from the Njombe ecotype. Two percent of the genetic diversity was due to differences among ecotypes, and the rest was due to differences among individuals within the ecotypes. Despite the overall low genetic differentiation, both fragment and sequencing analyses depicted a high allelic and genetic variability across 10 chicken ecotypes. These results therefore, underscore the importance of establishing appropriate conservation and management strategies to capitalize on observed variability and maintain genetic flexibility across diverse production environments.

Seven Japanese Herbals Prolonged Cardiac Allograft Survival.

Japanese herbal medicines have long been used as alternative therapy because of their immunomodulatory effects. In recent years, use herbal medicines is rapidly increasing worldwide. In this study, we investigated the effect of 17 components of traditional Japanese herbal medicines on alloimmune responses in a murine model of cardiac allograft transplantation. Fully vascularized heterotopic hearts from C57BL/6 donors were transplanted into CBA mice by using microsurgical techniques. Artemisiae capillaris herba (Inchinko) was given to CBA recipients at a dosage of 1 g/kg/day from the day of transplantation until 7 days afterward. The other 16 components were given at a dosage of 2 g/kg/day for the same time period. Naïve CBA mice rejected C57BL/6 cardiac grafts acutely (median survival time [MST] of 7 days). CBA transplant recipients given 2 g/kg/day of Glycyrrhizae radix (Kanzou), Poria sclerotium (Bukuryo), Pinellia tuber (Hange), Cnidii rhizome (Senkyu), Paeoniae radix (Shakuyaku), and Scutellariae radix (Ogon) had prolonged C57BL/6 allograft survival significantly (MSTs were 18, 18, 17, 14, 12, and 12 days, respectively). Moreover, CBA transplant recipients given 1g/kg/day of Artemisiae capillaris herba had prolonged C57BL/6 allograft survival (MST >100 days); however, none of other 10 components prolonged allograft survival. In conclusion, administration of 7 components of traditional Japanese herbal medicines might induce prolongation of fully major histocompatibility complex-mismatched cardiac allografts.

The Effects of Curcuma longa L., Purple Sweet Potato, and Mixtures of the Two on Immunomodulation in C57BL/6J Mice Infected with LP-BM5 Murine Leukemia Retrovirus.

The immune response is stimulated to protect the body from external antigens and is controlled by several types of immune cells. In the present study, the immunomodulatory effects of Curcuma longa L., purple sweet potato, and mixtures of the two (CPM) were investigated in C57BL/6 mice infected with LP-BM5 murine leukemia virus (MuLV). Mice were divided into seven groups as follows: normal control, infected control (LP-BM5 MuLV infection), positive control (LP-BM5 MuLV infection+dietary supplement of red ginseng 300 mg/kg body weight), the original powder of C. longa L. (C; LP-BM5 MuLV infection+dietary supplement of C 189 mg/kg body weight), the original powder of purple sweet potato (P; LP-BM5 MuLV infection+dietary supplement of P 1811 mg/kg body weight), CPM Low (CPL; LP-BM5 MuLV infection+CPM 2 g/kg body weight), and CPM High (CPH; LP-BM5 MuLV infection+CPM 5 g/kg body weight). Dietary supplementation lasted for 12 weeks. Dietary supplementation of CPM inhibited LP-BM5 MuLV-induced lymphadenopathy and splenomegaly and inhibited reduction of messenger RNA (mRNA) expression of major histocompatibility complex (MHC) I and II. Moreover, CPM reduced the decrease in T- and B cell proliferation, reduced the population of CD4(+)/CD8(+) T cells, and remedied the unbalanced production of T helper-1 (Th1)/T helper-2 (Th2) cytokines in LP-BM5 MuLV-infected mice. In addition, CPM inhibited reduction of phagocytosis in peritoneal macrophages and decreased serum levels of immunoglobulin A (IgA), immunoglobulin E (IgE), and immunoglobulin G (IgG). These results suggest that CPM had a positive effect on immunomodulation in C57BL/6 mice induced by LP-BM5 leukemia retrovirus infection.

Extracts of Tectona grandis and Vernonia amygdalina have anti-Toxoplasma and pro-inflammatory properties in vitro.

Tectona grandis (teak) and Vernonia amygdalina (bitter leaf) are plants used in traditional medicine in West Africa. In this study, we tested ethanolic and hydro-ethanolic extracts of bark and leaves of T. grandis and ethanolic extract of leaves of V. amygdalina for their inhibitory effect on Toxoplasma gondii, a protozoan parasite responsible for toxoplasmosis. Ethanolic extract of V. amygdalina leaves had proportional contents of phenols, tannins, flavonoids, and polysaccharides. This extract presented the highest efficacy against T. gondii, the lowest cytotoxicity to mammalian cells, but moderate anti-oxidant activity compared to other plant extracts. Ethanolic extract of T. grandis bark also had elevated anti-T. gondii activity, low cytotoxicity on mammalian cells, and one of the highest anti-oxidant activities. However, the phytochemical content of this extract was not very different from the hydro-ethanolic extract, which had no anti-T. gondii activity. In addition, ethanolic extract of V. amygdalina leaves, but not of T. grandis bark, significantly increased the production of TNF-α and NO by antigen-presenting cells. Both extracts had the tendency to decrease expression of major histocompatibility complex molecules at the surface of antigen-presenting cells, while they did not modulate the percentage of apoptotic cells. A study of signalling pathways would help to determine the mechanisms of action of these plant extracts.

TITLE: Les extraits de Tectona grandis et de Vernonia amygdalina ont des propriétés anti-Toxoplasma et pro-inflammatoires in vitro. ABSTRACT: Tectona grandis (teck) et Vernonia amygdalina sont des plantes utilisées dans la médecine traditionnelle en Afrique de l'Ouest. Dans cette étude, l'effet inhibiteur d'extraits éthanoliques et hydro-éthanoliques d'écorce et de feuilles de T. grandis et de l'extrait éthanolique des feuilles de V. amygdalina a été étudié sur Toxoplasma gondii, un parasite protozoaire responsable de la toxoplasmose. L'extrait éthanolique des feuilles de V. amygdalina avait des quantités équivalentes de phénols, tanins, flavonoïdes et polysaccharides. Cet extrait présentait la plus grande efficacité contre T. gondii, la plus faible cytotoxicité vis-à-vis de cellules de mammifères, mais une activité anti-oxydante moyenne comparée aux autres extraits de plantes. L'extrait éthanolique d'écorce de T. grandis avait aussi une activité anti-T. gondii élevée, une faible cytotoxicité vis-à-vis des cellules de mammifères et l'une des activités anti-oxydantes les plus élevées. Cependant, le contenu phytochimique de cet extrait n'était pas très différent de l'extrait hydro-éthanolique qui n'avait pas d'activité anti-T. gondii. De plus, l'extrait éthanolique des feuilles de V. amygdalina, mais pas de l'écorce de T. grandis, augmentait significativement la production de TNF-α et de NO par les cellules présentatrices d'antigènes. Les deux extraits avaient tendance à diminuer l'expression des molécules du complexe majeur d'histocompatibilité à la surface des cellules présentatrices d'antigènes alors qu'ils ne modulaient pas le pourcentage de cellules apoptotiques. L'étude des voies de signalisation permettrait de comprendre les mécanismes d'action de ces extraits de plantes.

Induction of Regulatory CD4+ Cells and Prolongation of Fully Major Histocompatibility Complex Mismatched Murine Cardiac Allograft by Shigyakusan.

Shigyakusan (also known as Tsumura Japan [TJ]-35) is composed of peony, bitter orange, licorice, and Bupleuri radix is used for cholecystitis and gastritis as an anti-inflammatory agent. We investigated the effect of TJ-35 on alloimmune response in a murine heart transplantation model. CBA mice that underwent transplantation of a C57BL/6 (B6) heart were assigned to four groups: no treatment, TJ-35-exposed, each component-exposed, or each component missing-exposed. The four groups above each received oral administration of TJ-35, each component, or TJ-35 with each component missing from the day of transplantation until 7 days, respectively. Untreated CBA recipients rejected B6 cardiac grafts acutely (median survival time [MST], 7 days). TJ-35-exposed CBA recipients had significantly prolonged B6 allograft survival (MST, 20.5 days). However, MSTs of CBA recipients that had been administered each component and TJ-35 with each component missing did not reach that of TJ-35-exposed recipients. Adoptive transfer of CD4+ splenocytes from TJ-35-exposed primary allograft recipients resulted in significant prolonged allograft survival in naïve secondary recipients (MST, 63 days). Flow cytometry studies showed that the percentage of CD4+CD25+Foxp3+ cell population was increased in TJ-35-exposed CBA recipients. In conclusion, TJ-35-induced prolongation of fully allogeneic cardiac allografts and may generate regulatory CD4+CD25+Foxp3+ cells in our model. The effect seemed to require all components of TJ-35.

Enhancing Radiation Therapy Through Cherenkov Light-Activated Phototherapy.

PURPOSE: This work investigates a new approach to enhance radiotherapy through a photo therapeutic agent activated by Cherenkov light produced from the megavoltage photon beam. The process is termed Radiotherapy Enhanced with Cherenkov photo-Activation (RECA). RECA is compatible with various photo-therapeutics, but here we focus on use with psoralen, an ultraviolet activated therapeutic with extensive history of application in superficial and extracorporeal settings. RECA has potential to extend the scope of psoralen treatments beyond superficial to deep seated lesions. METHODS AND MATERIALS: In vitro studies in B16 melanoma and 4T1 murine breast cancer cells were performed to investigate the potential of RT plus RECA versus RT alone for increasing cytotoxicity (local control) and increasing surface expression of major histocompatibility complex I (MHC I). The latter represents potential for immune response amplification (increased antigen presentation), which has been observed in other psoralen therapies. Cytotoxicity assays included luminescence and clonogenics. The MHC I assays were performed using flow cytometry. In addition, Cherenkov light intensity measurements were performed to investigate the possibility of increasing the Cherenkov light intensity per unit dose from clinical megavoltage beams, to maximize psoralen activation. RESULTS: Luminescence assays showed that RECA treatment (2 Gy at 6 MV) increased cytotoxicity by up to 20% and 9.5% for 4T1 and B16 cells, respectively, compared with radiation and psoralen alone (ie, Cherenkov light was blocked). Similarly, flow cytometry revealed median MHC I expression was significantly higher in RECA-treated cells, compared with those receiving radiation and psoralen alone (approximately 450% and 250% at 3 Gy and 6 Gy, respectively, P << .0001). Clonogenic assays of B16 cells at doses of 6 Gy and 12 Gy showed decreases in tumor cell viability of 7% (P = .017) and 36% (P = .006), respectively, when Cherenkov was present. CONCLUSION: This work demonstrates for the first time the potential for photo-activation of psoralen directly in situ, from Cherenkov light generated by a clinical megavoltage treatment beam.

The effect of feed supplementation with a copper-glycine chelate and copper sulphate on selected humoral and cell-mediated immune parameters, plasma superoxide dismutase activity, ceruloplasmin and cytokine concentration in broiler chickens.

The varied bioavailability and different effects of organic forms of copper on the immune system of poultry have prompted the search for new feed additives based on copper compounds containing modified chelate complexes. The aim of the study was to determine the effect of inorganic and organic forms of copper on selected parameters of the cellular and humoral immune response in broiler chickens by determining the percentages of CD3+ CD4+ , CD3+ CD8+ and CD25+ lymphocytes, cells with MHC Class II expression, and BU-1+ cells, as well as the concentrations of SOD, IL-2, IL-10 and TNF-α in the peripheral blood. The experiments were conducted using 500 one-day-old Ross 308 roosters divided into five groups. Cu was added in inorganic form (CuSO4 ), in inorganic form with the addition of phytase (CuSO4 + F), in organic form in combination with glycine (Cu-Gly) and in organic form in combination with glycine and a phytase supplement (Cu-Gly+F). The results of the study indicate an increase in the percentage of CD3+ CD4+ and CD3+ CD8+ T lymphocytes, CD25+ T cells, and cells expressing MHC class II molecules, and in the concentration of ceruloplasmin, activity of superoxide dismutase and the concentration of IL-2 in the groups that received copper, particularly copper-glycine chelates. Based on the study, we can conclude that supplementation of poultry feed with copper chelates activates mainly the Th1 cellular immune response and the response of peripheral blood T lymphocytes. Furthermore, it promotes secretion of cytokines, which are involved in potentiation and regulation of the immune response in birds.

Identification and characterization of CD4+ T cell epitopes on manganese transport protein C of Staphylococcus aureus.

Manganese transport protein C (MntC) of Staphylococcus aureus represents an excellent vaccine-candidate antigen. The important role of CD4+ T cells in effective immunity against S. aureus infection was shown; however, CD4+ T cell-specific epitopes on S. aureus MntC have not been well identified. Here, we used bioinformatics prediction algorithms to evaluate and identify nine candidate epitopes within MntC. Our results showed that peptide M8 emulsified in Freund's adjuvant induced a much higher cell-proliferation rate as compared with controls. Additionally, CD4+ T cells stimulated with peptide M8 secreted significantly higher levels of interferon-γ and interleukin-17A. These results suggested that peptide M8 represented an H-2d (I-E)-restricted Th17-specific epitope.

Identification of MHC Haplotypes Associated with Drug-induced Hypersensitivity Reactions in Cynomolgus Monkeys.

Drug-induced hypersensitivity reactions can significantly impact drug development and use. Studies to understand risk factors for drug-induced hypersensitivity reactions have identified genetic association with specific human leukocyte antigen (HLA) alleles. Interestingly, drug-induced hypersensitivity reactions can occur in nonhuman primates; however, association between drug-induced hypersensitivity reactions and major histocompatibility complex (MHC) alleles has not been described. In this study, tissue samples were collected from 62 cynomolgus monkeys from preclinical studies in which 9 animals had evidence of drug-induced hypersensitivity reactions. Microsatellite analysis was used to determine MHC haplotypes for each animal. A total of 7 haplotypes and recombinant MHC haplotypes were observed, with distribution frequency comparable to known MHC I allele frequency in cynomolgus monkeys. Genetic association analysis identified alleles from the M3 haplotype of the MHC I B region (B*011:01, B*075:01, B*079:01, B*070:02, B*098:05, and B*165:01) to be significantly associated (χ2 test for trend, p < 0.05) with occurrence of drug-induced hypersensitivity reactions. Sequence similarity from alignment of alleles in the M3 haplotype B region and HLA alleles associated with drug-induced hypersensitivity reactions in humans was 86% to 93%. These data demonstrate that MHC alleles in cynomolgus monkeys are associated with drug-induced hypersensitivity reactions, similar to HLA alleles in humans.

A Systems Approach to Understand Antigen Presentation and the Immune Response.

The mammalian immune system has evolved to respond to pathogenic, environmental, and cellular changes in order to maintain the health of the host. These responses include the comparatively primitive innate immune response, which represents a rapid and relatively nonspecific reaction to challenge by pathogens and the more complex cellular adaptive immune response. This adaptive response evolves with the pathogenic challenge, involves the cross talk of several cell types, and is highly specific to the pathogen due to the liberation of peptide antigens and their presentation on the surface of affected cells. Together these two forms of immunity provide a surveillance mechanism for the system-wide scrutiny of cellular function, environment, and health. As such the immune system is best understood at a systems biology level, and studies that combine gene expression, protein expression, and liberation of peptides for antigen presentation can be combined to provide a detailed understanding of immunity. This chapter details our experience in identifying peptide antigens and combining this information with more traditional proteomics approaches to understand the generation of immune responses on a holistic level.

Vitamin D3 administration to MS patients leads to increased serum levels of latency activated peptide (LAP) of TGF-beta.

BACKGROUND: Deficiency of vitamin D is an environmental risk factor for MS. Vitamin D has immunomodulatory effects, including promotion of T-cell differentiation into T-regulatory cells, which produces regulatory cytokines including TGF-ß. Increasing serum vitamin D levels have been associated with decreased disease activity in MS patients, but there are only few studies concerning the immunological effects of vitamin D supplementation in MS. In this study we investigated the effect of weekly supplementation of vitamin D3 or placebo on serum levels of multiple cytokines in patients with relapsing remitting MS. METHODS: The study was conducted on the patient cohort of the Finnish Vitamin D study. All patients were using IFN-beta-1b and were randomized to add-on treatment with either cholecalciferol 20,000 IU/week or placebo. Concentrations of LAP (TGF-ß), INF-γ, IL-17A, IL-2, IL-10, IL-9, IL-22, IL-6, IL-13, IL-4, IL-5, IL-1ß and TNF-α were determined at screening and at 12 months using commercial fluorescent bead immunoassay kits. RESULTS: LAP (TGF-ß) levels increased significantly in the vitamin D treated group from a mean of 47 (SE 11) pg/ml to 55 (SE 14) pg/ml in 12 months (p-value=0.0249). Placebo treatment had no significant effect on LAP levels. The levels of the other cytokines did not change significantly in either group. CONCLUSIONS: We showed increased serum latency activated peptide (LAP) of TGF-ß levels in MS patients treated with vitamin D3. The immune regulatory effects of TGF-beta may play a role in the improved MRI outcomes that we observed earlier in the vitamin D treated group of patients.

Bovine Neonatal Pancytopenia is a heritable trait of the dam rather than the calf and correlates with the magnitude of vaccine induced maternal alloantibodies not the MHC haplotype.

Bovine Neonatal Pancytopenia (BNP), a bleeding syndrome of neonatal calves, is caused by alloantibodies absorbed from the colostrum of particular cows. A commercial BVD vaccine is the likely source of alloantigens eliciting BNP associated alloantibodies. We hypothesized that the rare occurrence of BNP in calves born to vaccinated dams could be associated with genetic differences within dams and calves. We found that the development of BNP within calves was a heritable trait for dams, not for calves and had a high heritability of 19%. To elucidate which genes play a role in the development of BNP we sequenced candidate genes and characterized BNP alloantibodies. Alloantigens present in the vaccine have to be presented to the dam's immune system via MHC class II, however sequencing of DRB3 showed no differences in MHC class II haplotype between BNP and non-BNP dams. MHC class I, a highly polymorphic alloantigen, is an important target of BNP alloantibodies. Using a novel sequence based MHC class I typing method, we found no association of BNP with MHC class I haplotype distribution in dams or calves. Alloantibodies were detected in both vaccinated BNP and non-BNP dams and we found no differences in alloantibody characteristics between these groups, but alloantibody levels were significantly higher in BNP dams. We concluded that the development of BNP in calves is a heritable trait of the dam rather than the calf and genetic differences between BNP and non-BNP dams are likely due to genes controlling the quantitative alloantibody response following vaccination.

Potentiation of pH-sensitive polymer-modified liposomes with cationic lipid inclusion as antigen delivery carriers for cancer immunotherapy.

Cationic lipid-incorporated liposomes modified with pH-sensitive polymers were prepared by introducing 3, 5-didodecyloxybenzamidine as a cationic lipid to egg yolk phosphatidylcholine liposomes modified with 3-methylglutarylated hyperbranched poly(glycidol) (MGlu-HPG) as a pH-sensitive polymer. These liposomes were stable at neutral pH, but were destabilized below pH 6.0 because MGlu-HPG changed its characteristics from hydrophilic to hydrophobic in response to the pH decrease. Cationic lipid inclusion improved their pH sensitivity at weakly acidic pH and association of liposomes with murine dendritic cell (DC) lines. Cationic lipid-incorporated liposomes delivered entrapped ovalbumin (OVA) molecules not only to cytosol but also to endosome/lysosome. Treatment with cationic lipid-incorporated liposomes induced up-regulation of antigen presentation-involved molecules on DCs, the promotion of cytokine production, and antigen presentation via both major histocompatibility complex (MHC) class I and II molecules. Especially, antigen presentation via MHC class II was promoted by cationic lipid inclusion, which might correspond to efficient endosome/lysosome delivery of OVA. Subcutaneous administration of OVA-loaded cationic lipid-incorporated liposomes induced antigen-specific antibody production in serum and Th1-dominant immune responses in the spleen. Furthermore, administration of the cationic lipid-incorporated liposomes to mice bearing E.G7-OVA tumor more significantly reduced the tumor volume than liposomes without cationic lipids. Therefore, cationic lipid inclusion into pH-sensitive polymer-modified liposomes, which can achieve both efficient antigen intracellular delivery and activation of antigen presenting cell, is an effective approach to develop antigen carriers for efficient cancer immunotherapy.