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Supplemental Digital Content is available in the text., Adoptive transfer of T cells engineered with a chimeric antigen receptor (CAR) is deemed as the silver bullet to overcome the barriers of solid tumor treatment; however, the therapeutic application against solid tumors faces major challenges largely owing to the complex heterogeneity and immunosuppressive microenvironment of solid tumors. Preclinical development of CAR-T-cell products necessitates an appropriate animal model for the evaluation and improvement of their therapeutic capacities. Patient-derived xenograft (PDX) resembles real patients in several ways, and may serve as an attractive alternative to generate and evaluate the efficacy of CAR-T-cell products. In this study, we established and characterized a PDX mouse model implanted with colorectal cancer (CRC) xenograft. Human epidermal growth factor receptor 2 (HER2) expression in CRC specimens was detected by immunohistochemistry. The fragments of patient tumors were subcutaneously implanted into immunodeficient NOD-NPG mice after surgery. Furthermore, HER2-specific CAR-T cells were engineered and tested in our model to show their effectiveness in tumor clearance. Adoptive transfer of HER2-specific CAR-T cells resulted in the regression or even elimination of CRC xenograft and protection of relapse from rechallenged colon cancer tissue in PDX model. Significant survival advantage was achieved in these mice as compared with those transplanted with green fluorescent protein-T cells. Thus, this study showed that CAR-T-cell treatment may be a promising approach for solid tumor clearance and that the PDX model may be useful to evaluate the effects of CAR-T cells.

Chimeric antigen receptor (CAR) T cell therapy remains relatively ineffective against solid tumors due to inadequate infiltration and in vivo expansion of CAR-T cells. Unlike hematological malignancies, solid tumors have vascular barriers that hinder CAR-T cells from reaching the tumor site. Here, we demonstrated that combretastatin A-4 phosphate (CA4P), a vascular disrupting agent (VDA), can significantly improve the infiltration ability of CAR-T cells in solid tumors as evidenced by elevated levels of IFN-γ. Moreover, combined treatment with CA4P and CAR-T cells greatly increased the therapeutic efficiency of the CAR-T cells in subcutaneous ovarian cancer mouse xenograft models and patient-derived xenograft (PDX) models of colon and ovarian carcinoma. Our findings highlight CA4P as an effective antitumor agent candidate for combination with CAR-T cells in clinical applications to treat solid tumors.

Avian chimeras have been shown to be very useful tools for studying embryonic development. Blastodermal cells form stage X embryos and primordial germ cells (PGCs) are extensively used in chimera production. Intraspecies and interspecies germline chimeras have been successfully produced by transferring blastodermal cells or PGCs into recipient blastoderm at stage X or into the early embryonic blood. The long-term culture system established using pluripotent cells from birds provides an alternative method for preserving the endangered species source and facilitates the manipulation of avian transgenesis. A high rate of donor cell-derived progeny production has always been pursued by germline chimera studies. However, the rate of progeny production was unstable. Different methods have been used in an attempt to improve the efficiency of germline chimera production. Several methods have been shown to greatly enhance the efficiency of donor cell-derived progeny production. In this review, we focus on the strategies used for germline chimera production in birds and discuss the factors and measures affecting the efficiency of obtaining donor cell-derived progeny.

In the past, several strategies have been used to generate transgenic birds. The most successful method has proven to be injection of lentiviral vector into the subgerminal cavity of the newly laid egg. In this study, we directly injected lentiviral vector into the blood vessel of HH13-15 quail embryos to produce transgenic chimeras. In the manipulated, hatched birds, the green fluorescent protein (GFP) gene driven by a cytomegalovirus (CMV) promoter was extensively expressed. All tissues analyzed were GFP-positive, and gonad cells from some of the manipulated embryos expressed GFP. The semen genome of 21.4% of mature male birds was determined to be GFP-positive by PCR, indicating these male birds were transgenic chimeras.

The purpose of this study was to produce quail-duck chimeras by transferring stage X blastoderm cells and to detect the distribution of donor cells in heterogeneous embryos using PCR. Four experimental groups were made by transferring different amounts of quail blastoderm cells into duck recipients. In early embryonic stages, donor cells labeled with PKH26 fluorescent dye were observed in the head, neural tube and gonads by fluorescent microscopy. A total of 194 duck recipient embryos were injected and 93 survived to hatch. The average hatching rate was 48% (93/194); the hatching rate showed a significant difference among all the groups (P < 0.05). Sixteen somatic chimeras were obtained, 10 of which had black feathers derived from the donor quail. The PCR results showed that donor cells were distributed in various tissues and organs of the phenotypic chimeras. This is the first report on producing Japanese quail-Peking duck chimeras by transferring quail blastoderm cells into the subgerminal cavity of the duck. This technique will provide a basis for the investigation of fertilization barriers in interspecies germline chimeras and will aid conservation of endangered wild birds.

The production of quail–Peking duck, Peking duck–quail and Maya duck–chicken chimeras by transferring donor (quail, Peking duck and Maya duck) blastoderm cells into the subgerminal cavity of the recipient (Peking duck, quail and chicken) eggs is described. Production of chimeras by transferring blastoderm cells into the recipient embryo should provide a basis for the investigation of fertilisation barriers in interspecies germline chimeras. It might help to protect endangered birds, contribute to further understanding of physiology and embryonic development in poultry, and provide a technical method for poultry transgenics.

The response of Peking ducks to supplements of Sophy β-glucan was studied. A total of 160 healthy 1-d-old mixed-sex ducklings were randomly allocated to 3 groups: Sophy β-glucan (n = 80), bacitracin zinc (n = 40), and control (n = 40), which received the same antibiotics-deficient diet supplemented with 1% β-glucan, 5% bacitracin zinc, or nothing, respectively. During 2 mo of the study, growth performance, carcass composition, and meat quality of Peking ducks were evaluated. Additionally, a separate immunological study was conducted with a total of 105 healthy male Peking ducks in 7 groups (n = 15) and immunized with different doses of β-glucan (0, 0.5, 2.5, 12.5, and 62.5 μg/duck) and BSA (200 μg/duck). Blood was taken for detection of anti-BSA-IgG antibody and peripheral blood mononuclear cells proliferation assays. Groups subjected to different dietary treatments showed almost no differences in growth performance and slaughter traits except breast muscle percentage and intestinal length. These 2 indicators were significantly higher in the bacitracin zinc group than in the control and β-glucan groups (P < 0.05). Similarly, chemical compositions, fatty acids, and amino acids of breast muscle were not significantly influenced by the diet. Ducks immunized with Sophy β-glucan did not have enhanced level of anti-BSA-IgG antibodies but had significant peripheral blood mononuclear cells proliferation compared with unchallenged ducks (P < 0.01). With an increase in the glucan concentration, the proliferative responses approximately linearly increased. These findings indicate that 1% Sophy glucan did not improve duck growth performance, carcass composition, and meat quality significantly under the conditions of the present experiment and mainly had regulatory or enhancing properties on poultry nonspecific cellular immunity.

In the past, several strategies have been used to generate transgenic birds. The most successful method has proven to be injection of lentiviral vector into the subgerminal cavity of the newly laid egg. In this study, we directly injected lentiviral vector into the blood vessel of HH13-15 quail embryos to produce transgenic chimeras. In the manipulated, hatched birds, the green fluorescent protein (GFP) gene driven by a cytomegalovirus (CMV) promoter was extensively expressed. All tissues analyzed were GFP-positive, and gonad cells from some of the manipulated embryos expressed GFP. The semen genome of 21.4% of mature male birds was determined to be GFP-positive by PCR, indicating these male birds were transgenic chimeras.

The purpose of this study was to produce quail-duck chimeras by transferring stage X blastoderm cells and to detect the distribution of donor cells in heterogeneous embryos using PCR. Four experimental groups were made by transferring different amounts of quail blastoderm cells into duck recipients. In early embryonic stages, donor cells labeled with PKH26 fluorescent dye were observed in the head, neural tube and gonads by fluorescent microscopy. A total of 194 duck recipient embryos were injected and 93 survived to hatch. The average hatching rate was 48% (93/194); the hatching rate showed a significant difference among all the groups (P0.05). Sixteen somatic chimeras were obtained, 10 of which had black feathers derived from the donor quail. The PCR results showed that donor cells were distributed in various tissues and organs of the phenotypic chimeras. This is the first report on producing Japanese quail-Peking duck chimeras by transferring quail blastoderm cells into the subgerminal cavity of the duck. This technique will provide a basis for the investigation of fertilization barriers in interspecies germline chimeras and will aid conservation of endangered wild birds.

Ovarian cortical tissues of various ages of chicken were grafted underneath chorioallantoic membrane (CAM) of 6-d-old chicken embryo and the grafts were collected on d 0, 2, 4, 8, or 10. The tissue sections were prepared to examine the development of follicles in grafts and the proliferative ability of follicles was examined by immunohistochemistry analysis of proliferating cell nuclear antigen. The results indicated that the development of follicles in cultured chicken ovarian cortical pieces progressed smoothly and slowly similar to those in vivo, and the environment of chicken embryo was more suitable for the synchronous development of oocyte and follicular cells in the larger follicles. Meanwhile, the synchronous development could be more easily achieved in the chicken ovarian tissues of 12- to 15-wk chicken. Neither oocyte nor granulosa cells but the thecal cells of follicles developed in grafts of 13- to 15-d chicken within 10 d of culturing in ovo, and the CAM can ease follicular atresia in chicken ovarian grafts of 12- to 15-wk chicken. The proliferative performance of follicles in the grafts was not influenced by the environment of chicken embryo. These results indicated that the chicken embryo has the ability to support slow development of primordial and primary follicles in grafted ovarian cortical tissues of chickens of different ages. The CAM system may also prove useful for the study of early follicle development with further information.

Ghrelin is an acylated peptide and an endogenous ligand for the growth hormone secretagogue receptor (GHS-R), and stimulates growth hormone release and food intake in mammals. Peking duck is a very fast growing species of poultry. Although the sequence and structure of ghrelin have recently been determined, the expression of ghrelin in Peking duck has not been studied. Here, we investigated the tissue expression and distribution of ghrelin by RT-PCR and immunohistochemistry, respectively, in Peking duck at different stages of development. Ghrelin mRNA expression was mainly detected in the proventriculus and proventriculus–gizzard junction. It was first expressed, but weakly, on embryonic day 14 (E14); the expression increased by embryonic day 21 (E21), and was maintained at high levels between post-hatching-day 1 (P1) and post-hatching-day 60 (P60). Weak expression of ghrelin mRNA was also found in the gizzard and duodenum. In the gastrointestinal tract of growing Peking duck in P60, the largest number of ghrelin-ip cells was detected in the epithelium of the compound tubular glands in the proventriculus and the next largest number was in the proventriculus–gizzard junction. Very few ghrelin-ip cells were located in the epithelium of the simple tubular glands adjacent to the gizzard. No ghrelin-ip cells were observed elsewhere in the gastrointestinal tract. Ghrelin-ip cells were found in embryos as early as day E21; at the same time, the compound tubular glands in the proventriculus had formed. The numbers of ghrelin-ip cells on P1 were similar to those of E21 embryos. However, on P60, high numbers of strongly stained ghrelin-ip cells were found to be scattered in the epithelium of the compound tubular glands in the proventriculus. The density of ghrelin-ip cells (cells/mm 2 ) in the proventriculus on P60 was significantly greater than those of P1 and E21 embryos. These results demonstrate that ghrelin is expressed in the Peking duck gastrointestinal tract, especially in the proventriculus, from mid–late-stage embryos to growing period and suggested an involvement of ghrelin in the development and biology of the gastrointestinal tract of the Peking duck.