Genetic Links between Reproductive Traits and Amino Acid Pairwise Distances of Swine Leukocyte Antigen Alleles among Mating Partners in Microminipigs.

Previously, we found that a greater dissimilarity in swine leukocyte antigen (SLA) class I and class II alleles between mating partners resulted in increased farrowing rates in a highly inbred population of Microminipigs (MMPs). In this follow-up study, we have analyzed the effects of dissimilarity in SLA alleles between mating partners for seven different reproductive traits, including litter size and the number of stillborn and live or dead weaned piglets. We determined the relationships among reproductive traits within each mating event and the amino acid distances of SLA alleles as markers of diversity between mating partners. Our results indicate that mating partners with greater amino acid pairwise genetic distances in the SLA-1 class I gene or DQB1 class II gene alleles were associated with significantly larger litter sizes and higher numbers of live piglets at birth and weaning. Also, partners with greater pairwise distances in the SLA-2 class I gene alleles exhibited fewer pre-weaning deaths. These findings suggest that the dissimilarity in SLA class I and class II alleles between mating partners may affect not only farrowing rates but also other key reproductive traits such as litter size and improved piglet survival rates. Consequently, SLA alleles could serve as valuable genetic markers for selecting mating partners in breeding programs and for conducting epistatic studies on various reproductive traits in MMPs.

Genetic Association between Farrowing Rates and Swine Leukocyte Antigen Alleles or Haplotypes in Microminipigs.

We have previously reported specific swine leukocyte antigen (SLA) haplotype associations with significant effects on several reproduction performance traits in a highly inbred miniature pig population of Microminipigs (MMPs). In this study, to clarify the effects on farrowing rates of SLA similarity between mating partners in the MMP population, we compared the farrowing rates as a measure of reproductive success after 1063-cumulative matings among the following three groups of mating partners: (1) completely sharing SLA class I or class II haplotypes or alleles between partners (CS), (2) only one sharing the haplotypes or alleles (OS), and (3) non-sharing the haplotypes or alleles (NS). Average farrowing rates in CS groups consisting of completely sharing SLA class II haplotypes or DRBI and DQB1 alleles were lowest in the three groups. Moreover, lower farrowing rates were indicated in mating pairs with smaller amino acid pairwise genetic distances of SLA-1, SLA-3, DRB1 and DQB1 alleles between the pairs. These results suggested that the dissimilarity of SLA class I and class II alleles between mating partners markedly improved reproductive performance; therefore, SLA alleles or haplotypes are potentially useful genetic markers for the selection of mating pairs in breeding programs and epistatic studies of reproductive traits of MMPs.

Stillbirth rates and their association with swine leucocyte antigen class II haplotypes in Microminipigs.

OBJECTIVE: Microminipig (MMP) is a miniature pig with an extra small body size for experimental use. In the present study, the occurrence of stillbirths and their genetic association with swine leukocyte antigen (SLA) class II haplotypes were evaluated in a population of MMPs. METHODS: The occurrences of stillbirth and genetic association with SLA class II haplotypes using 483 stillborn and 2,246 live piglets, and their parents were compared among the three groups of newborn piglet litters; an all stillborn (AS) group consisting of only stillborn piglet litters, a partial stillborn (PS) group consisting of stillborn and live piglet litters, and an all alive (AA) group consisting of only live piglet litters. RESULTS: The incidence of stillborn piglets was 483/2,729 (17.7%). Distributions of litter sizes, numbers of stillborn piglets in a litter, parities, and gestation periods were distinct among the three groups. The frequencies of low resolution haplotype (Lr)-0.7 or Lr-0.23 were higher in the AS group than in the PS or AA groups. In sires, the frequency of Lr-0.7 associated with the AS group was significantly higher in the AS group than with the AA group. In dams, the frequency of Lr-0.23 was significantly higher in the AS group than in the PS or AA groups, whereas the frequency of Lr-0.7 was not significantly different. CONCLUSION: The incidence of stillborn piglets in MMPs appears to be higher than those in other pig breeds. Several traits related with stillbirths such as the number of stillborn piglets and parities of the AS group were different from those of the PS and AA groups. Specific SLA class II haplotypes were associated significantly with a high incidence of stillbirths and could be used as genetic markers to adopt breeding strategies to lower the rate of stillbirth in MMPs.

HER2-antigen-specific humoral immune response in breast cancer lymphocytes transplanted in hu-PBL hIL-4 NOG mice.

The status of humoral immunity of cancer patients is not clear compared to cellular immunity because the ability of specific antibody production is difficult to analyze in vitro. We previously developed a humanized mouse model to evaluate antigen-specific antibody production by transplanting human peripheral blood mononuclear cells (PBMCs) into NOG-hIL-4-Tg mice (hu-PBL hIL-4 NOG). In this study, these mice were transplanted with PBMCs derived from breast cancer patients (BC) and immunized with a human epidermal growth factor receptor 2 (HER2) peptide, CH401MAP, to analyze humoral immunity of BCs. The hu-PBL hIL-4 NOG mice recapitulated immune environment of BCs as the ratio of CD8+/CD4+T cells was lower and that of PD-1 + T cells was higher compared to healthy donors (HDs). Diverse clusters were detected in BC-mouse (BC-M) plasma components involving immunoglobulins and complements unlike HD-M, and there was a significant diversity in CH401MAP-specific IgG titers in BC-M. The number of B cell clones producing high CH401MAP-specific IgG was not increased by immunization in BC-M unlike HD-M. These results demonstrated that the humoral immunity of BCs appeared as diverse phenotypes different from HDs in hu-PBL hIL-4 NOG mice, which may provide important information for the study of personalized medicine.

Preparation and characterization of monoclonal antibodies recognizing two CD4 isotypes of Microminipigs.

Cluster of differentiation 4 (CD4) molecule expressed on the leukocytes is known to function as a co-receptor for class II major histocompatibility complex (MHC) binding to T cell receptor (TCR) on helper T cells. We previously identified two CD4 alleles (CD4.A and CD4.B) in a Microminipig population based on nucleotide sequencing and PCR detection of their gene sequences. However, CD4.B protein expression was not examined because of the unavailability of a reactive antibody to a CD4.B epitope. In this study, we have produced two swine-specific monoclonal antibodies (mAbs) against CD4.B molecules, one that recognizes only CD4.B (b1D7) and the other that recognizes both the CD4.A and CD4.B alleles (x1E10) and that can be used to distinguish CD4 T cell subsets by flow cytometry and immunohistochemistry. Using these two mAbs, we identified CD4.A and CD4.B allele-specific proteins on the surface of CD4.A (+/+) and CD4.B (+/+) T cells at a similar level of expression. Moreover, stimulation of peripheral blood mononuclear cells (PBMCs) derived from CD4.A (+/+) and CD4.B (+/+) swine with toxic shock syndrome toxin-1 (TSST-1) in vitro similarly activated both groups of cells that exhibited a slight increase in the CD4/CD8 double positive (DP) cell ratio. A large portion of the DP cells from the allelic CD4.A (+/+) and CD4.B (+/+) groups enhanced the total CD4 and class I swine leukocyte antigen (SLA) expression. The x1E10 mAb delayed and reduced the TSST-1-induced activation of CD4 T cells. Thus, CD4.B appears to be a functional protein whose expression on activated T cells is analogous to CD4.A.

Syntheses and Functional Studies of Self-Adjuvanting Anti-HER2 Cancer Vaccines.

The 9-mer peptide MFCH401 (N: 165-173: DTILWKDIF), which is located in the extracellular domain of HER2, has been predicted to be a novel epitope. Self-adjuvanting anti-HER2 vaccine constructs were designed and synthesized via covalently attaching MFCH401 or its linear tandem repeats (2×MFCH401, 3×MFCH401) to a lipopeptide Pam3 CSK4 via iterative condensation reaction. The in vivo results showed the Pam3 CSK4 -MFCH401 vaccine construct can induce higher antibody titers of IgG and IgM than those of other conjugates, and the analysis of changes in plasma cytokines level indicate the activation of Th1 cells and NK cells. In addition, the Pam3 CSK4 -MFCH401 vaccine conjugate induced a specific immune response to HER2-overexpressing human BT474 cells. Our data clearly indicated that MFCH401 is a promising epitope; moreover, its linear tandem repeats were unsuitable for anticancer vaccine design when conjugating with Pam3 CSK4 , which provided useful evidence for developing further anti-HER2 cancer vaccines.

Genetic Association between Swine Leukocyte Antigen Class II Haplotypes and Reproduction Traits in Microminipigs.

The effects of swine leukocyte antigen (SLA) molecules on numerous production and reproduction performance traits have been mainly reported as associations with specific SLA haplotypes that were assigned using serological typing methods. In this study, we intended to clarify the association between SLA class II genes and reproductive traits in a highly inbred population of 187 Microminipigs (MMP), that have eight different types of SLA class II haplotypes. In doing so, we compared the reproductive performances, such as fertility index, gestation period, litter size, and number of stillbirth among SLA class II low resolution haplotypes (Lrs) that were assigned by a polymerase chain reaction-sequence specific primers (PCR-SSP) typing method. Only low resolution haplotypes were used in this study because the eight SLA class II high-resolution haplotypes had been assigned to the 14 parents or the progenitors of the highly inbred MMP herd in a previous publication. The fertility index of dams with Lr-0.13 was significantly lower than that of dams with Lr-0.16, Lr-0.17, Lr-0.18, or Lr-0.37. Dams with Lr-0.23 had significantly smaller litter size at birth than those with Lr-0.17, Lr-0.18, or Lr-0.37. Furthermore, litter size at weaning of dams with Lr-0.23 was also significantly smaller than those dams with Lr-0.16, Lr-0.17, Lr-0.18, or Lr-0.37. The small litter size of dams with Lr-0.23 correlated with the smaller body sizes of these MMPs. These results suggest that SLA class II haplotypes are useful differential genetic markers for further haplotypic and epistatic studies of reproductive traits, selective breeding programs, and improvements in the production and reproduction performances of MMPs.

Expression of glucocorticoid receptor shows negative correlation with human B-cell engraftment in PBMC-transplanted NOGhIL-4-Tg mice.

The humanized mouse system is a promising tool for analyzing human immune responses in vivo. Recently, we developed a new humanized mouse system using the severely immunodeficient NOD/Shi-scid-IL2rγnull (NOG)-hIL-4-Tg mouse, which enabled us to evaluate the human humoral immune response after peripheral blood mononuclear cell (PBMC) transplantation. However, the mechanism by which hIL-4 enhances antigen-specific IgG production in these mice is not clear. In this study, we analyzed the relationship between human lymphocyte subsets and the expression level of the glucocorticoid receptor (GR) to clarify the humoral immune condition in human PBMC-transplanted NOG-hIL-4 mice. The results showed that the human GR mRNA level was significantly lower in NOG-hIL-4-Tg splenocytes than in conventional NOG splenocytes after immunization. Whereas no obvious difference of the proportion of T helper-cell subsets was observed between the NOG and NOG-hIL-4-Tg mouse strains, the B-cell proportion and antigen-specific IgG concentration in plasma showed strong negative correlations with the GR mRNA level. These results suggest that the GR expression level was changed in PBMCs in the humanized NOG-hIL-4-Tg mice, which may support B-cell survival and function in the mouse system.

B-cell populations are expanded in breast cancer patients compared with healthy controls.

BACKGROUND: Historically, humoral immunity was considered unimportant in anti-tumor immunity, and the differentiation and anti-tumor activity of B cells in breast cancer are poorly understood. However, it was recently discovered that B cells participate in tumor immunity through both antibody production and immunosuppressive mechanisms. We analyzed the expression of B-cell differentiation markers in detail using fluorescence-activated cell sorting to investigate the relationship between B-cell subsets and breast cancer etiology. METHODS: Blood samples were taken from breast cancer patients and healthy donors, and peripheral blood mononuclear cells were collected. B cells at various stages of differentiation were identified by the expression of combinations of the cell surface markers CD5, CD19, CD21, CD24, CD27, CD38, CD45, and IgD. Statistical analysis of the proportions of each B-cell subtype in the different patient groups was then performed. RESULTS: Twenty-seven breast cancer patients and 12 controls were considered. The proportion of total B cells was significantly higher in cancer patients than in controls (11.51 ± 2.059 vs 8.905 ± 0.379%, respectively; p = 0.001). Breast cancer patients were then classified as High-B or Low-B for further analysis. A significantly higher proportion of memory B cells was found in the High-B group than in the Low-B or control groups (p = 0.003 and p = 0.043, respectively). CONCLUSIONS: Breast cancer patients generally have a higher proportion of B cells than healthy controls, but this is highly variable. Analysis of the major B-cell surface markers indicates that memory B cells in particular are significantly expanded, or more robust, in breast cancer patients.

NOG-hIL-4-Tg, a new humanized mouse model for producing tumor antigen-specific IgG antibody by peptide vaccination.

Immunodeficient mice transplanted with human peripheral blood mononuclear cells (PBMCs) are promising tools to evaluate human immune responses to vaccines. However, these mice usually develop severe graft-versus-host disease (GVHD), which makes estimation of antigen-specific IgG production after antigen immunization difficult. To evaluate antigen-specific IgG responses in PBMC-transplanted immunodeficient mice, we developed a novel NOD/Shi-scid-IL2rγnull (NOG) mouse strain that systemically expresses the human IL-4 gene (NOG-hIL-4-Tg). After human PBMC transplantation, GVHD symptoms were significantly suppressed in NOG-hIL-4-Tg compared to conventional NOG mice. In kinetic analyses of human leukocytes, long-term engraftment of human T cells has been observed in peripheral blood of NOG-hIL-4-Tg, followed by dominant CD4+ T rather than CD8+ T cell proliferation. Furthermore, these CD4+ T cells shifted to type 2 helper (Th2) cells, resulting in long-term suppression of GVHD. Most of the human B cells detected in the transplanted mice had a plasmablast phenotype. Vaccination with HER2 multiple antigen peptide (CH401MAP) or keyhole limpet hemocyanin (KLH) successfully induced antigen-specific IgG production in PBMC-transplanted NOG-hIL-4-Tg. The HLA haplotype of donor PBMCs might not be relevant to the antibody secretion ability after immunization. These results suggest that the human PBMC-transplanted NOG-hIL-4-Tg mouse is an effective tool to evaluate the production of antigen-specific IgG antibodies.

High dose of antibiotic colistin induces oligomerization of molecular chaperone HSP90.

Colistin is an antimicrobial cationic peptide that belongs to the polymyxin family. Colistin was clinically used for the treatment of gram-negative infections but fell out of favour because of its significant side effects including neurotoxicity and nephrotoxicity. More recently, colistin has been regarded as one of the important options for nosocomial infections caused by multidrug resistant bacteria. Mechanisms of both the side effect onset of the drug and the side effect reduction are yet to be elucidated. In this study, we identified the specific binding protein of colistin using an affinity column chromatography. Colistin binds to the molecular chaperone HSP90. Although colistin slightly suppressed the chaperone activity of HSP90, there are no effects on the ATPase activity for a low concentration of colistin. Interestingly, colistin-induced aggregation of HSP90 via the N-domain. As for the cell viability of the SHSY5Y cell, the cell viability decreased to approximately 80% by the colistin 300 µM. However, the cell viability recovered to approximately 100% by adding ATP dosage. The same result was obtained by dot blot assay using anti-HSP90 antibody. Our results may help to understand the side effect mechanism of colistin.

Production of a Locus- and Allele-Specific Monoclonal Antibody for the Characterization of SLA-1*0401 mRNA and Protein Expression Levels in MHC-Defined Microminipigs.

The class I major histocompatibility complex (MHC) presents self-developed peptides to specific T cells to induce cytotoxity against infection. The MHC proteins are encoded by multiple loci that express numerous alleles to preserve the variability of the antigen-presenting ability in each species. The mechanism regulating MHC mRNA and protein expression at each locus is difficult to analyze because of the structural and sequence similarities between alleles. In this study, we examined the correlation between the mRNA and surface protein expression of swine leukocyte antigen (SLA)-1*0401 after the stimulation of peripheral blood mononuclear cells (PBMCs) by Staphylococcus aureus superantigen toxic shock syndrome toxin-1 (TSST-1). We prepared a monoclonal antibody (mAb) against a domain composed of Y102, L103 and L109 in the α2 domain. The Hp-16.0 haplotype swine possess only SLA-1*0401, which has the mAb epitope, while other haplotypes possess 0 to 3 SLA classical class I loci with the mAb epitopes. When PBMCs from SLA-1*0401 homozygous pigs were stimulated, the SLA-1*0401 mRNA expression level increased until 24 hrs and decreased at 48 hrs. The kinetics of the interferon regulatory transcription factor-1 (IRF-1) mRNA level were similar to those of the SLA-1*0401 mRNA. However, the surface protein expression level continued to increase until 72 hrs. Similar results were observed in the Hp-10.0 pigs with three mAb epitopes. These results suggest that TSST-1 stimulation induced both mRNA and surface protein expression of class I SLA in the swine PBMCs differentially and that the surface protein level was sustained independently of mRNA regulation.

Differentiation ability of multipotent hematopoietic stem/progenitor cells detected by a porcine specific anti-CD117 monoclonal antibody.

CD117 is a cytokine receptor expressed on the surface of hematopoietic stem cells with a likely role in cell survival, proliferation and differentiation. In order to study the differentiation activity of porcine CD117 hematopoietic cells in vitro and in vivo we prepared an anti-swine CD117 Mab (2A1) with high specificity for flow-cytometrical analysis. The 2A1 Mab did not recognize mouse or human mast cells suggesting that 2A1 is species-specific. Swine bone marrow (BM) CD117+ cells differentiated in vitro mainly into erythroid and monocyte lineages in the methylcellulose-based colony assay. When the swine BM CD117+ cells were transplanted in vivo into immunodeficient NOG (NOD/SCID/IL-2gc-null) mice, a significant amount of swine CD45+ leukocytes, including CD3 positive T cells, were developed in the mice. These results revealed that the swine BM CD117+ cells possess hematopoietic stem/progenitor activity and when monitored in immunodeficient mice or in vitro they can develop into lymphoid, erythroid, and myeloid cells efficiently with the new monoclonal antibody.