Anti-human leukocyte antigens and anti-major histocompatibility complex class I-related chain A antibody expression in kidney transplantation during a four-year follow-up

Background Humoral immunity is an important factor for long-term survival of renal allograft. Here we performed a four-year follow-up to explore the clinical significance of monitoring anti-human leukocyte antigens （HLA） and anti-major histocompatibility complex class I-related chain A （MICA） antibody expression after kidney transplantation. Methods We obtained serial serum samples from 84 kidney transplant patients over a four-year period. All patients were followed up at least 6 months after transplantation and had at least two follow-up points. Anti-HLA and anti-MICA antibody titres and serum creatinine （SCr） levels were evaluated at each follow-up. Patients were divided into 4 groups： HLA（＋） MICA（-）, HLA（-）MICA（＋）, HLA（＋）MICA（＋） and HLA（-）MICA（-）. The impact of post-transplant antibody level on kidney allograft function was evaluated. Results Antibodies were detected in 38.1% （32/84） of the renal allograft recipients. HLA, MICA and HLA＋MICA expression was observed in 18.89%, 14.44% and 5.93% of the recipients respectively. The most frequent anti-HLA and anti-MICA specific antibodies identified were All, A24, A29, A32, A33, A80; B7, B13, B37; DR17, DR12, DR18, DR52, DR53, DR1, DR4, DR9, DR51; DQ7, DQ4, DQ8, DQ2, DQ9, DQ5, DQ6 and MICA02, MICA18, MICA19, MICA07, MICA27. As the time after transplantation elapsed, more recipients developed de novo antibody expression. Total 11.91% （10/84） of the recipients had de novo antibody expression during the follow up. The average level of SCr and the percentage of recipients with abnormal allograft function were significantly higher in recipients with anti-HLA and/or anti- MICA antibody expression than those without. The appearance of anti-HLA and anti-MICA antibody expression always preceded the increase in SCr value. Conclusions Anti-HLA and anti-MICA antibody expression has predictive value for early and late allograft dysfunction. The presence of donor specific antibody is detrimental to graft function and graft survival.

Expression characteristics of major histocompatibility complex class I-related chain A antibodies and immunoadsorption effect in sensitized recipients of kidney transplantation

Background Sensitized recipients have a high risk of immunological graft loss due to hyperacute rejection and/or accelerated acute rejection. The presence of major histocompatibility complex class I-related chain A （MICA） antibodies has also been described associated with an increased rate of kidney-allograft rejection. The aim of this study was to describe the expression of MICA antibodies in sensitized recipients of renal transplantation and evaluate its influence on the kidney transplantation recipients.Methods A total of 29 sensitized recipients were included in this study. All patients received the MICA antibodies detection before and after protein A immunoadsorption. Panel reactive antibody （PRA）, HLA-matches, acute rejection and postoperative one to four-week serum creatinine level were also collected and analyzed, respectively. No prisoners were used in this study.Results Eight patients （27.6%） in all 29 sensitized recipients expressed the MICA antibodies but did not show higher acute rejection rate than the non-expressed patients （3/8, 37.5% vs. 8/21, 38.1%; P=1.000）. Recipients with PRA 〉40% showed higher expression levels of MICA antibodies than the recipients with PRA 〈40% （7/16, 43.8% vs. 1/13, 8.3%; P=0.044）. HLA mismatch did not have any effect on the expression of MICA antibodies （P=1.000）. MICA antibodies positive group had higher serum creatinine level than the control in postoperative one week （（135.4±21.4） μmol/L vs. （108.6±31.6） μmol/L, P=0.036）, but no significant difference in postoperative four weeks （（89.0±17.1） μmol/L vs. （77.1±15.9） μmol/L, P=0.089）. MICA antibodies decreased significantly after protein A immunoadsorption.Conclusions MICA antibodies increase in the sensitized recipients, which have significant effects on the function of aliograft in early postoperative period. Protein A immunoadsorption can decrease MICA antibodies effectively in sensitized recipients.

Antibodies against major histocompatibility complex class I-related chain A in transplant recipients

Objective To review the role of polymorphism of major histocompatibility complex class I-related chain A （MICA） gene and antibodies against MICA antigens in transplant immunology. Data sources The data used in this review were mainly from our own results and from the relevant English language literatures published from 1999 to 2010. Some data presented in this review are in press.Study selection Articles regarding MICA gene discovery and pioneering finding of antibodies against MICA antigen and allograft rejection were selected. This review chronicles the development of our understanding of the role that MICA antigens and antibodies may play in organ transplantation.Results Polymorphic glycoprotein MICA antigens were detected on freshly isolated human umbilical cord endothelial cells, but not on peripheral lymphocytes. Antibodies were found and typing of recipients and donors by sequencing the MICA alleles has established that de novo antibodies produced in kidney transplant recipients are directed at mismatched MICA epitopes and are associated with acute rejection and chronic transplant failure. The specificity of antibodies against the epitopes of MICA antigens were well characterized by donor MICA typing, single antigen array testing with antibody absorption and elution. Acute graft-versus-host disease was observed in stem-cell recipients who were mismatched for MICA.Conclusions Immunization against mismatched MICA epitopes encountered in donor organs after transplantation may result in antibodies against MICA alleles. Testing for MICA donor-specific antibodies （DSA） which are associated with early failure of kidney transplants may be helpful for identifying some of the targets of antibodies against antigens other than the human leukocyte antigen （HLA） and for improving transplantation outcome.

Recombination and mutation shape variations in the major histocompatibility complex

The major histocompatibility complex(MHC)is closely associated with numerous diseases,but its high degree of polymorphism complicates the discovery of disease-associated variants.In principle,recombination and de novo mutations are two critical factors responsible for MHC polymorphisms.However,direct evidence for this hypothesis is lacking.Here,we report the generation of fine-scale MHC recombination and de novo mutation maps of~5 Mb by deep sequencing(>100×)of the MHC genome for 17 MHC recombination and 30 non-recombination Han Chinese families(a total of 190 individuals).Recombination hotspots and Han-specific breakpoints are located in close proximity at haplotype block boundaries.The average MHC de novo mutation rate is higher than the genome-wide de novo mutation rate,particularly in MHC recombinant individuals.Notably,mutation and recombination generated polymorphisms are located within and outside linkage disequilibrium regions of the MHC,respectively,and evolution of the MHC locus was mainly controlled by positive selection.These findings provide insights on the evolutionary causes of the MHC diversity and may facilitate the identification of disease-associated genetic variants.

Major histocompatibility complex and mate choice in the polygynous primate:the Sichuan snub-nosed monkey(Rhinopithecus roxellana)

The highly polymorphic genes within the major histocompatibility complex(MHC)not only play a major role in immunity resistance,but also seem to provide hints for mate choice in some animal populations.In the pres­ent study we investigated MHC-related mate choice in a small natural population(group size 40-55 individuals)of a polygynous primate,the Sichuan snub-nosed monkey(Rhinopithecus roxellana).We found that there was no evidence either for MHC-disassortative mating,or for females to mate with males based on MHC hetero­zygosity or specific alleles.Nevertheless,of the 11 alleles identified,we found that the frequencies of 2 alleles,Rhro-DRB2(P<0.01)and Rhro-DRB5(P<0.05)were higher in offspring than in their parents.These findings suggest that MHC-DRB in this population of R.roxellana is unlikely to be associated with mating preferences.Limited female opportunities for mate choice are likely due,in part,to the harem breeding structure present in R.roxellana,and the relatively small number of resident adult males in our study band(N=4-6).In addition,we suggest that differences in the frequency of particular alleles across generations may be linked to parasite resis­tance in a fluctuating environment;however,confirmation of this finding requires further study.

Sequence variability analysis on major histocompatibility complex class Ⅱ DRB alleles in three felines

The variation of the exon 2 of the major histo-compatibility complex(MHC)class II gene DRB locus in three feline species were examined on clouded leopard(Neofelis nebulosa),leopard(Panthera pardus)and Amur tiger(Panthera tigris altaica).A pair of degenerated primers was used to amplify DRB locus covering almost the whole exon 2.Exon 2 encodes theβ1 domain which is the most vari-able fragments of the MHC class II molecule.Single-strand conformational polymorphism(SSCP)analysis was applied to detect different MHC class II DRB haplotypes.Fifteen recombinant plasmids for each individual were screened out,isolated,purified and sequenced finally.Totally eight distinct haplotypes of exon 2 were obtained in four individuals.With-in 237 bp nucleotide sequences from four samples,30 vari-able positions were found,and 21 putative peptide-binding positions were disclosed in 79 amino acid residues.The ratio of nonsynonymous substitutions(d_(N))was much higher than that of synonymous substitutions(d_(S)),which indicated that balancing selection probably maintain the variation of exon 2.MEGA neighbor joining(NJ)and PAUP maximum parsimo-ny(MP)methods were used to reconstruct phylogenetic trees among species,respectively.Results displayed a more close relationship between leopard and tiger;however,clouded leopard has a comparatively distant relationship form the other two.

Identification of TNFRSF1A as a novel regulator of carfilzomib resistance in multiple myeloma

Multiple myeloma(MM)is a hematological tumor with high mortality and recurrence rate.Carfilzomib is a new-generation proteasome inhibitor that is used as the first-line therapy for MM.However,the development of drug resistance is a pervasive obstacle to treating MM.Therefore,elucidating the drug resistance mechanisms is conducive to the formulation of novel therapeutic therapies.To elucidate the mechanisms of carfilzomib resistance,we retrieved the GSE78069 microarray dataset containing carfilzomib-resistant LP-1 MM cells and parental MM cells.Differential gene expression analyses revealed major alterations in the major histocompatibility complex(MHC)and cell adhesion molecules.The upregulation of the tumor necrosis factor(TNF)receptor superfamily member 1A(TNFRSF1A)gene was accompanied by the downregulation of MHC genes and cell adhesion molecules.Furthermore,to investigate the roles of these genes,we established a carfilzomib-resistant cell model and observed that carfilzomib resistance induced TNFRSF1A overexpression and TNFRSF1A silencing reversed carfilzomib resistance and reactivated the expression of cell adhesion molecules.Furthermore,TNFRSF1A silencing suppressed the tumorigenesis of MM cells in immunocompetent mice,indicating that TNFRSF1A may lead to carfilzomib resistance by dampening antitumor immunity.Furthermore,our results indicated that TNFRSF1A overexpression conferred carfilzomib resistance in MM cells and suppressed the expression of MHC genes and cell adhesion molecules.The suppression of MHC genes and cell adhesion molecules may impair the interaction between immune cells and cancer cells to impair antitumor immunity.Future studies are warranted to further investigate the signaling pathway underlying the regulatory role of TNFRSF1A in MM cells.

Next-generation Sequencing of MHC Class Ⅰ Genes Reveals Trans-species Polymorphism in Eutropis multifasciata and Other Species of Scincidae

The genes of the major histocompatibility complex(MHC) encode cell surface proteins that are essential for adaptive immunity. MHC genes show the most prominent genetic diversity in vertebrates,reflecting the adaptation of populations to their evolving environment, population survival and reproduction. In the present study, we used nextgeneration sequencing(NGS) to study the loci polymorphism of exon 3 of the MHC class Ⅰ genes in an ovoviviparous skink, the many-lined sun skink,Eutropis multifasciata and five other species of Scincidae, to quantify genetic variation. In addition,we genotyped the same MHC class Ⅰ genes of E.multifasciata using clone sequencing, to directly compare the effectiveness of both analytical techniques for MHC genotyping. NGS detected 20MHC class Ⅰ alleles in E. multifasciata, and 2 to 15 alleles in the other five Scincidae species. However,clone sequencing detected only 15 of those MHC class Ⅰ alleles in E. multifasciata. In addition, transspecies polymorphism of MHC class Ⅰ genes was studied by constructing a phylogenetic tree using the gene sequences obtained by NGS. Phylogenetic analysis revealed that MHC class I alleles were shared among different species of Scincidae with trans-species polymorphism, and did not exhibit specific genealogical inheritance. These results have important implications for understanding polymorphism interspecies diversity in the MHC genes of Scincidae, and the evolution of the MHC more broadly.

Development of a humanized HLA-A30 transgenic mouse model

Background:There are remarkable genetic differences between animal major histocompatibility complex(MHC)systems and the human leukocyte antigen(HLA)system.HLA transgenic humanized mouse model systems offer a much better method to study the HLA-A-related principal mechanisms for vaccine development and HLA-Arestricted responses against infection in human.Methods:A recombinant gene encoding the chimeric HLA-A30 monochain was constructed.This HHD molecule contains the following:α1-α2 domains of HLA-A30,α3 and cytoplasmic domains of H-2D~b,linked at its N-terminus to the C-terminus of humanβ2m by a 15-amino-acid peptide linker.The recombinant gene encoding the chimeric HLA-A30 monochain cassette was introduced into bacterial artificial chromosome(BAC)CH502-67J3 containing the HLA-A01 gene locus by Red-mediated homologous recombination.Modified BAC CH502-67J3 was microinjected into the pronuclei of wild-type mouse oocytes.This humanized mouse model was further used to assess the immune responses against influenza A virus(H1N1)pdm09 clinically isolated from human patients.Immune cell population,cytokine production,and histopathology in the lung were analyzed.Results:We describe a novel humanβ2m-HLA-A30(α1α2)-H-2D~b(α3 transmembrane cytoplasmic)(HHD)monochain transgenic mouse strain,which contains the intact HLA-A01 gene locus including 49 kb 5’-UTR and 74 kb 3’-UTR of HLA-A01*01.Five transgenic lines integrated into the large genomic region of HLA-A gene locus were obtained,and the robust expression of exogenous transgene was detected in various tissues from A30-18#and A30-19#lines encompassing the intact flanking sequences.Flow cytometry revealed that the introduction of a large genomic region in HLA-A gene locus can influence the immune cell constitution in humanized mice.Pdm09 infection caused a similar immune response among HLA-A30 Tg humanized mice and wild-type mice,and induced the rapid increase of cytokines,including IFN-γ,TNF-α,and IL-6,in both HLA-A30 humanized Tg mice and wild-type mice.The expression of HLA-A30 transgene was dramatically promoted in tissues from A30-9#line at 3 days post-infection(dpi).Conclusions:We established a promising preclinical research animal model of HLA-A30 Tg humanized mouse,which could accelerate the identification of novel HLA-A30-restricted epitopes and vaccine development,and support the study of HLA-A-restricted responses against infection in humans.

Stress increases MHC-I expression in dopaminergic neurons and induces autoimmune activation in Parkinson’s disease

The expression of major histocompatibility complex class I(MHC-I),a key antigen-presenting protein,can be induced in dopaminergic neurons in the substantia nigra,thus indicating its possible involvement in the occurrence and development of Parkinson’s disease.However,it remains unclear whether oxidative stress induces Parkinson’s disease through the MHC-I pathway.In the present study,polymerase chain reaction and western blot assays were used to determine the expression of MHC-I in 1-methyl-4-phenylpyridinium(MPP+)-treated SH-SY5Y cells and a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)-induced Parkinson’s disease mouse model.The findings revealed that MHC-I was expressed in both models.To detect whether the expression of MHC-I was able to trigger the infiltration of cytotoxic T cells,immunofluorescence staining was used to detect cytotoxic cluster of differentiation 8(CD8)+T cell infiltration in the substantia nigra of MPTP-treated mice.The results indicated that the presentation of MHC-I in dopaminergic neurons was indeed accompanied by an increase in the number of CD8+T cells.Moreover,in MPTP-induced Parkinson’s disease model mice,the genetic knockdown of endogenous MHC-I,which was caused by injecting specific adenovirus into the substantia nigra,led to a significant reduction in CD8+T cell infiltration and alleviated dopaminergic neuronal death.To further investigate the molecular mechanisms of oxidative stress-induced MHC-I presentation,the expression of PTEN-induced kinase 1(PINK1)was silenced in MPP+-treated SH-SY5Y cells using specific small interfering RNA(siRNA),and there was more presentation of MHC-I in these cells compared with control siRNA-treated cells.Taken together,MPP+-/MPTP-induced oxidative stress can trigger MHC-I presentation and autoimmune activation,thus rendering dopaminergic neurons susceptible to immune cells and degeneration.This may be one of the mechanisms of oxidative stress-induced Parkinson’s disease,and implies the potential neuroprotective role of PINK1 in oxidative stress-induced MHC-I presentation.All animal experiments were approved by the Southern Medical University Ethics Committee(No.81802040,approved on February 25,2018).

Immunity phenomena following olfactory ensheathing cell transplantation into experimental allergic encephalomyelitis rat brain

Olfactory ensheathing cells （OECs） can promote axonal regeneration and remyelination for the treatment of spinal cord injury. OECs can also treat experimental allergic encephalomyelitis （EAE）, but it remains unclear whether OECs might be rejected by the immune system in the brain including the destruction of the blood-brain barrier under inflammation, the release of inflammatory factors, the activation of local antigen-presenting cells （e.g., microglia cells） and antigen drainage. We found that OECs expressed major histocompatibility complex （MHC）-I molecules on the cell surface, barely expressed MHC-II, but MHC-II could be induced by interferon-v, suggesting that OECs have certain immunogenicity. When OECs were transplanted into normal animal brains, no OECs were phagocytosed by dendritic cells in the cervical lymph node, and OECs did not induce lymphocyte proliferation, which indicates that OECs share some immune privilege under normal conditions. However, OECs in the rat EAE brain were phagocytosed by dendritic cells in the cervical lymph node and enhanced lymphocyte proliferation. These findings suggest that OECs are rejected because of increased immunogenicity in EAE brain, and that brain inflammation, in particular activated dendritic cells, may be a prerequisite for rejecting OECs.

Sequence Comparison of MHCClassⅡβ(Exon 2)and Phylogenetic Relation-ship Between Poultry and Mammalian

A fragment spanning over exon 2 and intron 2 of major histocompatibility complex B-LB Ⅱ genes was amplified using PCR, cloned and sequenced in 13 individuals from eight Chinese indigenous chicken breeds and one introduced breed. Another 41 sequences of MHC class Ⅱ β from ten vertebrate species were cited from the NCBI GenBank. Thirteen new B-LB Ⅱ alleles were found in the chicken breeds sampled. Alignment of the exon 2 sequences revealed 91.1-97.8% similarity to each other within the chickens sampled, and the chickens shared 84.1-87.0% homology to Phasianus colchicus, 78.5-81.5% similarity to Coturnix japonica. The sequences in poultry showed 62.6-68.1% identity to HLA-DRB1, 50-61.5% similarity to DQB (HLA-, SLA- and H2-BB), 53.7-60% to HLA-DPB and 53.3-57.8% similarity to HLA-DOB. The frequency of nonsynonymous substitutions of nucleotide was higher than that of synonymous substitutions, and the frequencies of nonsynonymous and synonymous substitutions in poultry B-LB Ⅱ genes were lower than those observed in mammalian DRB1 and DQB1 genes. The deduced amino acid sequences of MHC class Ⅱ β1 domain exhibited extreme difference in conversed region and variable region patterns among the various species, but the two conserved cysteines forming disulfide-bond were shown consistent in poultry with that in mammalian species; and the carbohydrate attachment site was found more conserved in chicken, Homo sapiens, Bos taurus, Ovis aries and Capra hircus than in Sus scrofa and rodent animals. Compared with exon 2 of DQB1 genes of Homo sapiens, ruminant species and Sus scrofa, the differentia that the deletion of six nucleotides at position195 to 200 of exon 2 of DQB1 genes, and insertion of three nucleotides at position 247 to 249 of the exon 2 existed in rodent species were found, which led to the absence of three AA residues at position 65, 66, and 67 within β1 domain of DQB1 chain, and the insertion of one AA residue at position 85. The difference of the deletion of six nucleotides at position 72 to 77 of exon 2 of DPB1 genes was observed with Homo sapiens DQB1, which caused absence of three AA residues at position 24, 25, and 26 of β1 domain of DPB1 chain. The phylogenetic tree revealed that the B-LB Ⅱ sequences from poultry are not orthologous to the class Ⅱ MHC β-chain genes of mammalian species. The tree indicated that genetic evolutionary relationship of chickens with Phasianus colchicus was much closer than with Coturnix japonica, and the DQB and DPB clusters are more tightly related to each other than to the remaining clusters.

Cloning,characterization,and expression of a novel member of proteasomal subunits gene in turbot,Scophthalmus maximus

The proteasome is a large, polymeric protease complex responsible for the degradation of intracellular pro- teins and generation of peptides that bind to class I major histocompatibility complex （MHC） molecules. This study identified a new member of proteasomal subunits in turbots （Scophthalrnus rnaxirnus）. The full- length cDNA sequence of turbot proteasomal subunit was obtained. Sequence analysis indicated that its primary structure is highly similar to that of LMP7 from other vertebrates. The relationship between the turbot LMP7 expression and immune responses to pathogen infection was reported. Quantitative reverse transcriptase polymerase chain reaction showed that LMPTwas expressed differently in various tissues, with higher expression in the spleen, liver, muscle, and skin. The LMP7 expression was the highest at 96 h after challenge with lymphocyctis disease virus （LCDV） and at 12 h after challenge with Vibrio anguillarurn in the turbot liver, kidney, and spleen. Furthermore, the LMP7 expression distinctly increased in turbot kidney cells at 24 h after challenge with V. anguillarurn and at 96 h after challenge with LCDV. These results indicate that the turbot LMP7 protein participates in immune responses and may play a significant role in the immune process.

Induction of animal model of Graves' disease in BALB/c mice

Objective To construct an animal model of Graves’ disease(GD)by immunizing BALB/c mice with hM12 cells co-expressing major histocompatibility complex(MHC)class II molecules and human thyrotropin receptor(TSHR)molecules.Methods BALB/c mice in experimental group(H-2d)were immunized with hM12 cells intraperitoneally every 2 weeks for six times,while mice in control group were immunized with M12 cells.Five weeks later,the thyroids were histologically examined,and serum samples were tested for thyroid-stimulating antibodies(TSAb)and thyroid hormone levels.Results One BALB/c mouse in experimental group developed Graves’-like disease.Total T4 and T3 levels in this mouse were above the upper limit of normal,TSAb activity was displayed in its serum.The thyroid histologically showed the features of thyroid hyperactivity including thyrocyte hypercellularity and colloid ABSorption.None of control mice developed Graves’-like disease.Conclusion An animal model with some characteristics of human Graves’ disease was successfully induced and the model will facilitate studies aimed directly at understanding the pathogenesis of autoimmunity in Graves’ disease.

Antigen presenting cells may be able to distinguish between normal and radiatedSchistosoma japonicum cercaria:anin vitro observation

Objective： To observe the discrepancies of responses induced by Schistosoma japonicum （S. japonicum） normal cercaria antigen （NCA） and ultraviolet （UV） -radiation-attenuated cercaria antigen （UVACA） in an in vitro system. Methods： S. japonicum cercariae were collected and UVACA and NCA were prepared. Mouse macro- phage model cells （RAW 264.7） were treated with medium, NCA （40 μg/mL） or UVACA （40 μg/mL） in the presence or absence of recombinant mouse interferon gamma （rmIFN-γ; 4 ng/mL） for 48 h. Cell surface staining and flow cytometry were used to assess the major histocompatibility complex （MHC） Ⅱ expression, and data were expressed as mean fluorescence intensities （MFI）. Interleukin （IL） -10, IL-6 and prostaglandin E2 （PGE2） in cell culture supernatant were evaluated by commercial enzyme-linked immunosorbent assays. Results： NCA significantly suppressed IFN-7-induced MHC Ⅱ expression on RAW 264.7 cells. In the presence of 1FN-7, NCA significantly promoted IL-6, IL-10 and PGE2 secretion from RAW 264.7 cells. In the presence of IFN-γ, UVACA significantly promoted IL-10 but not IL-6 and PGE2 secretion from RAW 264.7 cells and showed no effect on IFN-γ-induced MHC Ⅱ expression. Compared with UVACA, NCA significantly suppressed IFN-γ-induced MHC Ⅱ expression and significantly promoted IL-6, PGE2 and IL-10 secretion from RAW 264.7 cells. Conclusion： RAW 264.7 cells respond differently to NCA and UVACA. NCA can significantly suppress IFN-γ-induced MHC Ⅱ expression and significantly promote IL-6, IL-10 and PGE2 secretion from RAW 264.7 cells compared with UVACA.

EXPRESSION OF IMMUNE-RELATED MOLECULES IN GLIOBLASTOMA MULTIFORM CELLS

Objective: To investigate the expression of immune- related molecules in glioblastoma multiform(GBM) cells. Methods: The expression of major histocompatibility complex (MHC), b2-microglobulin, Fas, CD80 and CD86 molecules on the surface of GBM cells were evaluated by flow cytometry. The expression of TAP-1, TAP-2 and Tapasin in the GBM cells were evaluated by RT-PCR method. Results: MHC class I, b2 microglobulin, TAP-1, TAP-2 and tapasin were expressed in most GBM cell lines. Except U87, there was no MHC class II molecule expression on any of the other GBM cell lines. Fas was expressed on all the GBM cell lines examined. Conclusion: The mechanism by which GBM escapes immune surveillance may involve down regulation of expression of MHC class I molecules and MHC class II molecules. MHC class I positive GBM may be the suitable target of immunotherapy.

Identification of BoLA DRB3.2 Alleles Present in White Fulani and Muturu Cattle Breeds

Cattle production is an important aspect of animal agriculture. Disease predisposition in cattle can lead to lowered productivity and poor animal welfare. To select and breed for the fittest cattle population, it is expedient that we understand the genetic basis of disease tolerance/resistance. Major histocompatibility complex (MHC) is a vital component of the immune system in vertebrates. Its genes are crucial determinants for immune response and resistance to infectious diseases. The bovine MHC is referred to as Bovine Lymphocyte antigen (BoLA) with its most functional and highly variable region located in the exon 2 (BoLA-DRB3.2). Over 100 alleles of BoLA-DRB3.2 have been identified in cattle and many studies have associated polymorphism in this region with disease resistance/susceptibility. In this study, we investigated the polymorphic nature of BoLA-DRB3.2 in the White Fulani and Muturu cattle breeds using a single PCR-sequence based typing. We identified 26 and 25 alleles in White Fulani and Muturu breeds, respectively, with only six alleles being mutual in the two breeds. Some of the alleles identified in this study have been noted as markers for disease status in cattle. BoLA-DRB3*014:01:01, BoLA-DRB3*011:01, and BoLA-DRB3*008:01 alleles have been associated with Bovine leukemia virus (BLV) resistance in cattle. BoLA-DRB3*014:01:01, BoLA-DRB3*001:01, and BoLA-DRB3*011:01 were linked with mastitis resistance in Japanese Holstein cows. While no inference can be drawn in terms of association with disease status, this study confirms the highly polymorphic and diverse nature of BoLA-DRB3 in White Fulani and Muturu cattle breeds.

Tumor antigen and MHC expression in glioma cells for immunotherapeutic interventions

AIM: To investigate the expression of tumor-antigens and major histocompatibility complex(MHC)-machinery components in glioblastoma multiforme cell lines flow cytometry staining methods were applied.METHODS: Ten GBM cell lines(three commercially available: U-87 MG, U-138-MG and GMS-10 as well as seven newly established cell lines from individual patients in low-passages: HROG02, HROG04, HROG05, HROG06, HROG10, HROG13 and HROG17) were analyzed for expression of(Ⅰ) general and(Ⅱ) GBMrelated tumor antigens as well as of(Ⅲ) components of the MHC machinery by flow cytometry.RESULTS: All cell lines expressed MHC class?Ⅰ?with seven out of the ten being HLA-A02 positive. Four of the seven primary cell lines additionally expressedMHC class Ⅱ in a constitutive manner. Of note, after interferon gamma(IFN-γ) treatment, all seven cell lines expressed MHC class Ⅱ. The tumor associated antigens(TAA) EGFR and survivin were expressed at high levels in all cell lines; whereas MART-1, RHAMM, WT-1 and IL-13Rα were expressed by at least half of the cell lines and HER2/neu, MAGE-1 and tyrosinase were expressed only by few cell lines. However, all cell lines expressed at least two of the candidate antigens included into this analysis.CONCLUSION: No obvious differences between commercially available and newly-established cell lines were observed. Thus, the latter in low-passages are interesting for(therapy-) screening and immunotherapeutic strategies.

Neutrophils in Atlantic salmon(Salmo salar L.)are MHC class II^(+)and secret IL-12p40 upon bacterial exposure

Antigen-presentation via major histocompatibility complex(MHC)to T cells is the key event to initiate adaptive immune responses.In teleosts,as in mammals,the main types of professional antigen-presenting cells(APCs)are dendritic cells(DCs),monocytes/macrophages,and B cells.In the current study,flow cytometry,immunostaining and qPCR have been used to show that neutrophils in the teleost fish Atlantic salmon(Salmo salar L.)have antigen-presenting properties.The neutrophils were positive for MHC class II,CD83 and CD80/86,and upon in vitro bacterial exposure,gene expression analysis of purified neutrophils showed that IL-12p40,which is essential for proliferation of naïve T cells,was highly upregulated at both 6 and 24 h post bacterial exposure.Based on presence of MHC class II and upregulation of molecules involved in antigen presentation and T cell activation,we suggest that neutrophils in Atlantic salmon have potential to function as professional APCs.This work makes an important basis for further exploring the potential of using neutrophils to develop new,targeted immunoprophylactic measures.

Identification of novel genes associated with atherosclerosis in Bama miniature pig

Background:Atherosclerosis is a chronic cardiovascular disease of great concern.However,it is difficult to establish a direct connection between conventional small animal models and clinical practice.The pig's genome,physiology,and anatomy reflect human biology better than other laboratory animals,which is crucial for studying the pathogenesis of atherosclerosis.Methods:We used whole-genome sequencing data from nine Bama minipigs to perform a genome-wide linkage analysis,and further used bioinformatic tools to filter and identify underlying candidate genes.Candidate gene function prediction was performed using the online prediction tool STRING 12.0.Immunohistochemistry and immunofluorescence were used to detect the expression of proteins encoded by candidate genes.Results:We mapped differential single nucleotide polymorphisms(SNPs)to genes and obtained a total of 102 differential genes,then we used GO and KEGG pathway enrichment analysis to identify four candidate genes,including SLA-1,SLA-2,SLA-3,and TAP2.nsSNPs cause changes in the primary and tertiary structures of SLA-I and TAP2 proteins,the primary structures of these two proteins have undergone amino acid changes,and the tertiary structures also show slight changes.In addition,immunohistochemistry and immunofluorescence results showed that the expression changes of TAP2 protein in coronary arteries showed a trend of increasing from the middle layer to the inner layer.Conclusions:We have identified SLA-I and TAP2 as potential susceptibility genes of atherosclerosis,highlighting the importance of antigen processing and immune response in atherogenesis.