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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.

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 were higher compared to healthy donor (HD). 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. (198)

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.

Because accumulation of mutations produces unique neoantigen patterns in cancer cells, it has been hypothesized that these patterns are recognized by the patients' immune system. Therefore, the development of cancer vaccines has been challenging owing to the possibility of an anti-cancer effect induced by the immune system against such neoantigens. However, it is difficult to develop effective vaccines because of the variety of mutations induced in cancer cells and human leukocyte antigen (HLA), which is predicted to present the neoantigen-derived peptides. Moreover, the activation of cancer-specific cytotoxic cells is inhibited by cancer cell immunoediting in each patient. Although cellular immunity can be analyzed ex vivo, there are no definite methods to evaluate humoral immunity. A humanized mouse model has been developed and used for evaluating the multipotency of hematopoietic stem cells. Presently, significant improvements in the humanized mouse model approach have partially recapitulated human humoral immunity in vivo, and human antibody production can be induced in the mouse model. These mice can be used to produce new cancer vaccines and to establish polypharmacy protocols in a preclinical model. In this review, we discuss the preclinical evaluation of the cancer vaccines using humanized mice transferred with patient-derived peripheral blood mononuclear cells (PBMCs) to advance a personalized medicine approach.

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. Electronic supplementary material The online version of this article (10.1007/s12282-017-0824-6) contains supplementary material, which is available to authorized users.

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.

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.

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.

Currently, many peptide vaccines are undergoing clinical studies. Most of these vaccines were developed to activate cytotoxic T cells; however, the response is not robust. Unlike vaccines, anti-cancer antibodies based on passive immunity have been approved as a standard treatment. Since passive immunity is more effective in tumor treatment, the evidence suggests that limited B cell epitope-based peptide vaccines may have similar activity. Nevertheless, such peptide vaccines have not been intensively developed primarily because humoral immunity is thought to be preferable to cancer progression. B cells secrete cytokines, which suppress immune functions. This review discusses the possibility of therapeutic antibody induction by a peptide vaccine and the role of active and passive B cell immunity in cancer patients. We also discuss the use of humanized mice as a pre-clinical model. The necessity of a better understanding of the activity of B cells in cancer is also discussed.

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.

Summary Microminipigs are extremely small-sized, novel miniature pigs that were recently developed for medical research. The inbred Microminipigs with defined swine leukocyte antigen (SLA) haplotypes are expected to be useful for allo- and xenotransplantation studies and also for association analyses between SLA haplotypes and immunological traits. To establish SLA-defined Microminipig lines, we characterized the polymorphic SLA alleles for three class I (SLA-1, SLA-2 and SLA-3) and two class II (SLA-DRB1 and SLA-DQB1) genes of 14 parental Microminipigs using a high-resolution nucleotide sequence-based typing method. Eleven class I and II haplotypes, including three recombinant haplotypes, were found in the offspring of the parental Microminipigs. Two class I and class II haplotypes, Hp-31.0 (SLA-1*1502–SLA-3*070102–SLA-2*1601) and Hp-0.37 (SLA-DRB1*0701–SLA-DQB1*0502), are novel and have not so far been reported in other pig breeds. Crossover regions were defined by the analysis of 22 microsatellite markers within the SLA class III region of three recombinant haplotypes. The SLA allele and haplotype information of Microminipigs in this study will be useful to establish SLA homozygous lines including three recombinants for transplantation and immunological studies.

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.

Background: Our previous predictive peptide binding studies indicated that a novel 20-mer multiple antigen peptide, CH401MAP, containing an anti-human epidermal growth factor receptor 2 (HER2) monoclonal antibody epitope (N163-182), may potentially bind to more than 95% of class I human leukocyte antigens (HLAs) and to 30-50% of class II HLAs expressed on peripheral blood mononuclear cells (PBMCs). In this study, CH401MAP was used for in vitro stimulation of PBMCs obtained from Japanese breast cancer patients, and anti-CH401MAP antibody secretion was evaluated. Methods: PBMCs of breast cancer patients were stimulated with CH401MAP peptide in vitro. Eight days after stimulation, the culture supernatants were collected and the anti-CH401MAP antibody levels were determined using enzyme-linked immunosorbent assay . The correlation of the antibody level and HER2 expression level after in vitro stimulation was also evaluated. Results: CH401MAP specific antibody was detected in the culture supernatants of patients’ PBMCs after in vitro culture, irrespective of the peptide stimulation. The antibody levels of the three patient’s groups were significantly higher than that of HD group. Significant correlation was not observed between specific antibody production and cancer progression . Conclusion: The PBMC of Japanese breast cancer patients possessed the potential of anti-CH401MAP antibody secretion. The antibody secretion level was significantly higher than that of HD. It is correlated with the expression of HER2 on the cancer tissues but not with the HER2 level in the sera of patients.